Boronate-Mediated Delivery of Molecules into Cells

ABSTRACT

Methods for enhancing cellular uptake of cargo molecules by boronating the cargo molecule, particularly with one or more phenylboronic acid groups. Cellular uptake includes at least partial uptake into the cytosol. Boronation includes ligating, crosslinking or otherwise bonding one or more phenylboronic acids substituted to contain a reactive group to a cargo molecule. Boronation also includes ligating, crosslinking or otherwise bonding a phenylboronated oligopeptide to a cargo molecule. The phenylboronate groups are optionally conjugated to the cargo molecule via linking moieties that can be selectively cleaved, such cleavable linkers can allow the phenylboronate groups to be removed from the cargo molecule after the boronated cargo molecule is introduced into the cell. The invention includes certain phenylboronates which are boronation reagents, certain boronated oligopeptides and certain boronated peptides and proteins. The invention also includes kits for enhancing cellular uptake of cargo molecules by boronation with one or more phenylboronates or boronated oligopeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application61/588,120, filed Jan. 18, 2012 which application is incorporated byreference herein in its entirety.

STATEMENT REGARDING U.S. GOVERNMENT FUNDING

This invention was made with government support under CA073808 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

The utility of many biologic drugs is limited by inefficient cellulardelivery [1]. Previous efforts to overcome this limitation have focusedlargely on the use of cationic domains, including peptidic cationicspecies (e.g., HIV-TAT, penetratin, and nonaarginine and more generallycell penetrating peptides (CPP)) or non-peptidic cationic species (e.g.,PAMAM dendrimers and polyethylimine), to enhance the attraction betweena chemotherapeutic agent and the anionic cell surface [2]. Naturalligands (e.g., folic acid, substance P, and the RGD tripeptide) havealso been used to facilitate cellular delivery by targeting agents tospecific cell-surface receptors [3]. Such methods have been applied, forexample, to delivery of peptide, proteins, nucleic acids and analogsthereof, reporters and labels, various pharmaceuticals and drugs andvarious small molecules as well as particles. Although some of thesemethods have had some success, there remains a need in the art foradditional delivery strategies.

The cell surface of many prokaryotic and eukaryotic cells is coated witha dense forest of polysaccharides known as the glycocalyx [4]. Targetingof therapeutic agents to the glycocalyx might enhance their cellulardelivery, as has been demonstrated with lectin conjugates [5]. Boronicacids readily form boronate esters with the 1,2- and 1,3-diols ofsaccharides [6], including those in the glycocalyx [7]. In addition,boronate groups are compatible with human physiology, appearing inchemotherapeutic agents and other remedies [8]. The present inventionrelates to the use of pendant boronic acids to mediate the delivery of acargo molecule into mammalian cells.

Boronic acids have been employed in the development of sensors forglycans [6] and in the development of boron neutron capture therapiesfor cancer treatment [24]. Boronic acids have also been reported asuseful in glucose sensors for the controlled release of insulin [26].Certain diboronic acid compounds have been reported useful asfluorescent sensors for certain cancer cells which express sialyl LewisX [7b].

Boronic acids have been reported useful in drug delivery applications,for example, to transport nucleotides and monosaccharides acrossliposomal bilayers [8a, 25], for labeling of liposomes for increasedaffinity for erythrocyte ghosts [7a] and for enhancement of PEI-DNAcomplexation for transfection reagents [8e].

U.S. Pat. No. 4,499,082 reports certain alpha-aminoboronic acid peptidesas reversible inhibitors of proteolytic enzymes. U.S. Pat. No. 6,018,020relates to boronated amino acid derivatives which are viral proteinaseinhibitors. U.S. Pat. Nos. 6,465,433; 6,548,668; and U.S. publishedapplication 2009/0325903 report certain amino acid and peptidyl boronicacids as inhibitors of proteasome function and more specifically asinhibitors of HIV replication in an animal. In each of these references,a carboxylic acid group of an amino acid is replaced with a boronic acidgroup (—B(OH)₂) or an ester thereof. Each of these references isincorporated by reference herein in its entirety for its description ofamino acids and peptidyl boronic acids and any description therein ofmethods of synthesis thereof.

U.S. published application 20110059040 relates to methods for inhibitingthe activity of a virus or bacterium by contacting the virus orbacterium with a polymer functionalized with at least one boronic acidmoiety, including one or more substituted or unsubstituted arylboronicacids, particularly phenylboronic acids. Certain multivalentbenzoboroxole functionalized polymers have been reported to inhibitmicrobicide entry [8b].

U.S. Pat. Nos. 5,594,111; 5,594,151; 5,623,055; 5,777,148; 5,744,627;5,837,878; and 6,156,884 relate to phenylboronic acid complexingreagents and complexes formed with such complexing reagents which arereported to be useful for preparation of bioconjugates. Similar reportsof phenyldiboronic acid complexing reagents and complexes formedtherewith are found in U.S. Pat. Nos. 6,075,126; 6,124,471; 6,462,179and 6,630,577. Each of these references is incorporated by referenceherein in its entirety for descriptions of phenylboronic acid compoundsand phenyldiboronic acid compounds and methods of synthesis thereof.

The physical properties including sugar binding properties of certainbenzoboroxoles and benzoxaborins have been studied [13, 22, 27].Syntheses of certain benzoboroxoles have been reported [30-36]. A reviewof applications of benzoboroxoles has been provided [28]. Thesereferences are incorporated by reference herein for methods of synthesisand sugar binding properties of benzoboroxoles.

The design, synthesis and screening of a library of peptidylbis(boroxoles) as receptors for the tumor marker TF-Antigen disaccharidein water has been reported [22]. The library components contained a freeamine, a certain PEG linker, two diaminoproponic acids residues to whichbenzoboroxole groups were attached separated by a central amino acid(where the library components were randomized with selected natural andnon-natural amino acids) and a capping group (where the librarycomponents were randomized with 20 selected capping groups). Thisreference is incorporated by reference herein for its description of thelibrary species and methods of synthesis of the library species. Anypeptidyl bis(boroxoles) noted in this reference can optionally beexcluded from the claims herein.

Gold electrodes activated by phenylboronic acid diazonium salts arereported for the reversible immobilization of eukaryotic cells [7c]. Anelectrochemical cell sensor for the determination of K562 leukemia cellsusing 3-aminophenylboronic acid (APBA)-functionalized multiwalled carbonnanotubes (MWCNTs) films is reported [7e]. K562 leukemia cells arereported to be bound to the APBA-functionalized MWCNTs film via boronicacid groups. This reference is incorporated by reference herein in itsentirety for descriptions of phenylboronic acid diazonium salts andsynthesis thereof.

SUMMARY OF THE INVENTION

The present invention relates to methods for enhancing cellular uptakeof a cargo molecule by boronating the cargo molecule, particularly withone or more phenylboronic acid groups. In a specific embodiment,cellular uptake includes at least partial uptake into the cytosol.Cellular uptake may be in vivo or in vitro. The method of the inventionis generally useful for the delivery of any desired molecule into a celland specifically includes nucleic acids and analogs thereof; nucleotidesand analogs thereof; peptides and proteins; drugs (e.g., anticancerdrugs, alkylating agents, antimetabolite, cytotoxic agents; antibiotics,and the like); reporter molecules or labels (e.g., fluorescent labels,isotopic labels, imaging agents, quantum dots, and the like). In aspecific embodiment, the cargo comprises a quantum dot carrying aminefunctionality. The cargo molecule can include combinations of thespecies listed above, wherein the species are bonded to each other,particularly where the species are covalently bonded to each other. Forexample, a cargo molecule may combine a peptide, such as a CPP or anuclear localizing signal with a nucleic acid, or combine a fluorescent,isotopic or other label with a nucleic acid and or peptide. In aspecific embodiment, the cargo is or comprises a molecule which affects,regulates or modulates gene expression in the cell, including a moleculewhich inhibits or decreases gene expression or a molecule whichinitiates or enhances gene expression. In a specie embodiment, the cargois a peptide or a protein, for example, an enzyme.

In one embodiment, the boronating method relates to forming one or morebonds between the cargo molecule and one or more phenylboronic acidgroups to boronate the cargo molecule. In this embodiment, one or morephenylboronic acids substituted to contain a reactive group as describedherein are reacted with one or more reactive groups of the cargomolecule. More specifically, the one or more bonds formed may becovalent bonds formed between one or more reactive groups on the cargomolecule and a reactive group on the one or more phenylboronic acidcompounds. The reaction of these reactive groups results in theformation of a linking moiety between the phenylboronate group and thecargo molecule. The linking moiety comprises the covalent bond or bondsnewly formed by reaction, can also comprise residual moieties from thereactive groups and can contain one or more selected spacer moieties.For example, the reactive groups on the cargo molecule or the reactivegroups on the phenylboronate compound can themselves be optionallyattached to the cargo molecule or the phenylboronate group,respectively, via a spacer moiety. In this case the linking moiety thatresults from reaction of the reactive groups contains any such spacingmoieties in the starting cargo molecule or phenylboronate compounds,bonds that are formed by reaction of reactive groups and any residue ofthe reactive groups. In a specific embodiment, the linking moietybetween the phenylboronate and the cargo molecule also contains a labelwhich functions to label the cargo molecule.

In a specific embodiment, the phenylboronate groups are conjugated tothe cargo molecule via linking moieties that can be selectively cleaved.In a more specific embodiment, phenylboronate groups can be cleaved fromthe cargo molecule after the boronated cargo molecule is introduced intothe cell. In a specific embodiment, the cleavable linker contains alatent reactive group, which is selectively activated to cleave thelinker and thereby cleave the phenylboronate(s) from the cargo molecule.In other specific embodiments, the linker is cleaved via selectiveactivation of a latent reactive group on the phenyl boronate whichfunctions to cleave the linker molecule and thereby cleave thephenylboronate(s) from the cargo molecule. In a specific embodiment, thephenylboronates are esterase releasable, e.g., the linker is cleavableby esterase action. In more specific embodiments, the linker iscleavable by a trimethyl lock mechanism. In other specific embodiments,the linker is a coumarin-based esterase releasable linker moiety.

In a specific embodiment, the linking moiety between the phenylboronateand the cargo molecule also contains a label which functions on bindingor ligation of the boronated oligopeptide to the cargo molecule to labelthe cargo molecule.

In general, the reactive groups of the cargo molecule and the phenylboronate are selected to achieve the desired linkage and for any desiredselectable cleavage, or more specifically to achieve a desired covalentlinkage which is optionally selectively cleavable and which is suitablefor the desired application of the boronated cargo molecule.

In another embodiment, the boronating method involves attaching aboronated amino acid or a boronated oligopeptide, which are substitutedwith one or more phenylboronate groups, to the cargo molecule. Theboronated amino acid or oligopeptide can be generated by formingcovalent bond(s) between one or more reactive groups on an amino acid oroligopeptide and a reactive group on one or more phenylboronic acidcompounds. The reaction of these reactive groups results in theformation of a linking moiety between the phenylboronate group and theamino acid or oligopeptide. As noted above, the linking moiety comprisesnewly formed bonds, particularly covalent bonds, and any residualmoieties from the reactive groups. Additionally, the reactive groups onthe amino acid, oligopeptide or the phenylboronate compound arethemselves optionally attached to the amino acid, oligopeptide orphenylboronate compound via a spacer moiety. In this case the linkingmoiety that results from reaction of the reactive groups contains anysuch spacer moieties in the starting amino acid, oligopeptide orphenylboronate compounds, bonds that are formed by reaction of reactivegroups and any residue of the reactive groups. The reactive groups ofthe oligopeptide may be those that occur naturally in amino acidstherein, may be those that occur in non-naturally occurring amino acidsintroduced into the oligopeptide by any art-known method or they mayresult from chemical modification of such naturally occurring reactivegroups as is known in the art as noted above.

Boronated amino acids and boronated oligopeptides may also becommercially available or be made by art-recognized methods. Theboronated amino acid or the boronated oligopeptide can be attached tothe cargo molecule to be boronated by any art-recognized method.

For example, a boronated amino acid or oligopeptide can be modified, forexample, to contain a ligand which selectively binds to the cargopeptide or protein, such as biotin or a derivative thereof such asbiocytin.

In another alternative method, the boronated oligopeptide can be coupledto the cargo molecule employing a crosslinking reagent which may contain2 or more reactive groups for crosslinking. Homobifunctional andheterobifunctional crosslinking reagents are particularly useful. Forexample, the boronated oligopeptide can be coupled to the cargo moleculeemploying a homobifunctional crosslinking reagent or aheterobifunctional crosslinking reagent.

More generally, heterofunctional crosslinking reagents may include aplurality of reactive groups which are the same and one or moredifferent reactive groups. Use of such heterofunctional crosslinkingreagents will allow, for example, linking of two or more phenylboronicacid groups to a single cargo molecule.

In another embodiment, boronation of a boronated oligopeptide forattachment to a cargo molecule or boronation of a cargo peptide orprotein can be accomplished by introduction of one or more boronatedamino acids into the peptide or protein. Boronated amino acids can beintroduced onto the peptide or protein directly by solid phase peptidesynthesis wherein one or more of the amino acid derivatives employed forpeptide synthesis are boronated to contain a phenylboronate group orbenzoboroxole group. Alternatively, a boronated oligopeptide can begenerated by solid phase peptide synthesis employing one or moreboronated amino acids as starting materials for peptide synthesis. Theboronated oligopeptide can be boronated cargo (where the cargo is theoligopeptide) or the boronated oligopeptide can thereafter be bound to,ligated to or crosslinked to the cargo which is to be boronated.

Boronated oligopeptides useful as boronated cargo or useful forboronating cargo can include those having 2-30 amino acids and morespecifically those having 5 to 20 amino acids. Boronated oligopeptidesinclude those where 60% or less, 50% or less, 40% or less, or 25% orless of the amino acids of the oligopeptide carry a phenylboronategroup. Boronated oligopeptides include those carrying 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 phenylboronic acid groups. Boronated oligopeptidesinclude those comprising one or more glutamic acids and/or asparticacids which are boronated with phenylboronate groups.

Amino acids boronated with phenylboronate or benzoboroxole are availablefrom art-known methods or are prepared as described above for theboronation of peptides and protein by reaction of a reactive group ofthe amino acid with a reactive group of a phenylboronate orbenzoboroxole compound. The phenylboronate or benzoboroxole groups isbonded to the amino acid via a linking moiety as described above whichcontains any bond resulting from reaction of the reactive groups, anyresidue of the reactive group, any spacer moieties in the starting aminoacid or phenylboronate or benzoboroxole compound and any optional label.

Boronated peptides, proteins and oligopeptides of this invention arealso optionally labeled for direct or indirect detection by anyart-known method. In specific embodiments, suitable labels includeisotopic labels, dyes, fluorescent or chemiluminescent groups ormoieties, radiolabels, haptens and the like. In a specific embodiment, alabel may be included in the linking moiety between at least onephenylboronate group and the peptide or protein. In specificembodiments, wherein cleavable linkers are employed, a label may beincluded in the linking moiety such that the label remains linked to thepeptide or protein on cleavage of the linker. In alternativeembodiments, wherein cleavable linkers are employed, a label may beincluded in the linking moiety such that the label is removed from thepeptide or protein on cleavage of the linker.

Reactive groups of the cargo peptides or proteins or of oligopeptides oramino acids used for boronation may be those that occur naturally inamino acids (e.g., proteinogenic amino acids) therein, may be those thatoccur in non-naturally occurring amino acids introduced into the peptideor protein by any art known method or they may result from chemicalmodification of such naturally-occurring or non-naturally-occurringreactive groups as is known in the art. The reactive groups of peptidesor proteins may, among others, be C-terminal carboxylate groups,N-terminal amine groups, and/or amine groups, sulfhydryl groups,hydroxyl groups or carboxylate groups of a side group of anynaturally-occurring amino acid of the peptide or protein. In specificembodiments, the reactive groups which are used for boronation are aminegroups and the boronation is facilitated by formation of an amide bond.The reactive groups of the peptide or protein may also, among others, beactivated ester groups, thioester groups, phosphinothiol ester groups,azide groups, aldehyde and/or ketone groups generated in or introducedinto the peptide or protein by any art-known method.

When the cargo molecule is a peptide or protein, boronation can beachieved, for example, by ligating a boronate oligopeptide to thepeptide or protein by any known method of peptide ligation, particularlyby the formation of an amide bond between the N- or C-termini of theoligopeptide and the peptide or protein. Alternatively, the boronatedoligopeptide can be modified, for example, to contain a ligand whichselectively binds to the cargo peptide or protein, such as biotin or aderivative thereof such as biocytin.

When the cargo is a nucleic acid or analog thereof, the reactive groupused to facilitate boronation may be any of those that occur naturallyin nucleic acids, and particularly any of those that occur in nucleosidebases as well as those that occur in non-naturally occurring nucleosidesor those that can be introduced into the nucleotide or nucleoside by anyart known method or they may result from chemical modification of suchnaturally-occurring or non-naturally-occurring reactive groups as isknown in the art. In specific embodiments, the reactive groups which areused for boronation are amine groups and the boronation is facilitatedby formation of an amide bond. The preferred reactive group forboronation of nucleic acids is an exo-amino group of the nucleic acid,e.g., an amino group of a nucleoside base, e.g., the amino groups ofcytosine, guanine, or adenine bases or analogs thereof (e.g.,isocytosine, isoguanine, diaminopurine or diaminopyridine, among others)which contain amino groups.

In specific embodiments, the invention provides boronation reagents,particularly those which are phenyl boronates or benzoboroxoles. Suchreagents can comprise reactive groups and/or latent reactive groups tofacilitate bonding to cargo molecules or to facilitate selectivecleavage of any linkers formed with cargo molecules. Such reagents canalso comprise spacer moieties (within linkers formed) or reporter orlabel moieties (optionally within linkers formed or otherwise formed inthe reagent.).

In specific embodiments, phenylboronic acid and benzoboroxole compoundsemployed for boronation in this invention have only one boronic acidgroup —B(OH)₂, —B(OH)— moiety or B atom in the compound. In specificembodiments, the phenyl ring of phenylboronic acids or benzoboroxole issubstituted with one or more electron withdrawing groups. Morespecifically the phenyl ring of phenylboronic acids, benzoboroxoles, orof the phenylboronic or benzoboroxole groups is substituted at the 2-and/or 4-ring positions (i.e., ortho or para positions) with respect tothe bond to the boron with one to four electron withdrawing groups. Inspecific embodiments, the phenylboronate is substituted at the 2-ringposition with a —(CR₇R₈)_(x)—OH group, where x is 1 or 2 and R₇ and R₈are as defined below, which respectively form a 5-member boroxole or a6-member oxaborin ring, where in specific embodiments each R₇ and R₈ isa hydrogen.

In specific embodiments, the cargo molecule is boronated to contain 4 ormore, 5 or more, 10 or more or 20 or more phenylboronate orbenzoboroxole groups. In more specific embodiments, the peptide orprotein is boronated to contain 2-10 phenylboronate or benzoboroxolegroups. In specific embodiments, a cargo molecule can optionally beboronated with two or more different phenylboronate or benzoboroxolegroups. In a specific embodiment, a peptide or protein is boronated withone or more phenylboronate or benzoboroxole groups that are all thesame.

In specific embodiments, the phenylboronic acids employed for boronationare selected from those having formula IA or IB:

(noting that IB is a benzoboroxole structure) and salts thereof,

where:

x is 1 or 2;

R₁-R₆ are independently selected from hydrogen, a straight-chain orbranched aliphatic group having 1-8 carbon atoms, an alicyclic group, anaryl group, a heterocyclic group, a heteroaryl group, a —CO₂R₁₀ group, a—O—CO—R₁₀ group, a —CON(R₁₂)₂ group, a —O—CON(R₁₂)₂ group; a —N(R₁₂)₂group, a —OR₁₀ group, a —(CH₂)_(m)—OH group, a —(CH₂)_(m)—N(R₁₂)₂ group,a halogen, a nitro group, a cyano group, a —SO₂—OR₁₀ group, -M, or twoadjacent R₂-R₆, together with the ring carbons to which they areattached, optionally form a 5-8-member alicyclic, heterocyclic, aryl orheteroaryl ring moiety, each of which groups or moieties is optionallysubstituted;

each R₇ and R₈ is independently selected from hydrogen or a C1-C3optionally substituted alkyl group;

wherein:

each R₁₀ is independently selected from hydrogen, a straight-chain orbranched aliphatic group having 1-8 carbon atoms, an alicyclic group, anaryl group, a heterocyclic group, or a heteroaryl group, each of whichgroups is optionally substituted;

each R₁₂ is independently selected from hydrogen, a straight-chain orbranched aliphatic group having 1-8 carbon atoms, an alicyclic group, anaryl group, a heterocyclic group, a heteroaryl group, or where two R₁₂together with the nitrogen to which they are attached can form a 5-8member heterocyclic or heteroaryl ring moiety, each of which groups ormoieties is optionally substituted;

m is an integer from 1-8;

M is a reactive group or a spacer moiety substituted with a reactivegroup for forming a bond to a cargo molecule and wherein at least one ofR₁-R₆ is M; and

wherein optional substitution is substitution by one or moresubstituents selected from halogen; an oxo group (═O), a nitro group; acyano group; a C1-C6 alkyl group; a C1-C6 alkoxy group; a C2-C6 alkenylgroup; a C2-C6 alkynyl group; a 3-7 member alicyclic ring, wherein oneor two ring carbons are optionally replaced with —CO— and which maycontain one or two double bonds; an aryl group having 6-14 carbon ringatoms; a phenyl group; a benzyl group; a 5- or 6-member ringheterocyclic group having 1-3 heteroatoms and wherein one or two ringcarbons are optionally replaced with —CO— and which may contain one ortwo double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O orS); a —CO₂R₁₃ group; —OCO—R₁₃ group; —CON(R₁₄)₂ group; —OCON(R₁₄)₂group; —N(R₁₄)₂ group; a —SO₂—OR₁₃ group, —OR₁₃ group, —(CH₂)_(m)—OR₁₃group, —(CH₂)_(m)—N(R₁₄)₂, where m is 1-8 and each R₁₃ or R₁₄ isindependently hydrogen; an unsubstituted C1-C6 alkyl group; anunsubstituted aryl group having 6-14 carbon atoms; an unsubstitutedphenyl group; an unsubstituted benzyl group; an unsubstituted 5- or6-member ring heterocyclic group, having 1-3 heteroatoms and wherein oneor two ring carbons are optionally replaced with —CO— and which maycontain one or two double bonds; or a heteroaryl group having 1-3heteroatoms (N, O or S) and in addition two R₁₄ together with thenitrogen to which they are attached can form a heterocyclic orheteroaryl ring moiety, each of which groups or moieties is optionallysubstituted;

each of which R₁₃ and R₁₄ groups is in turn optionally substituted withone or more unsubstituted C1-C3 alkyl groups, halogens, oxo groups (═O),nitro groups, cyano groups, —CO₂R₁₅ groups, —OCO—R₁₅ groups, —CON(R₁₆)₂groups, —OCO—N(R₁₆)₂ groups, —N(R₁₆)₂ groups, a —SO₂—OR₁₅ group, —OR₁₅groups, —(CH₂)_(m)—OR₁₅ groups, —(CH₂)_(m)—N(R₁₆)₂ where m is 1-8 andeach of R₁₅ and R₁₆ independently are hydrogen, an unsubstituted C1-C6alkyl group; an unsubstituted aryl group having 6-14 carbon ring atoms;an unsubstituted phenyl group; an unsubstituted benzyl group, anunsubstituted 5- or 6-member ring heterocyclic group having 1-3heteroatoms and wherein a ring carbon is optionally replaced with —CO—and which may contain one or two double bonds; or a heteroaryl grouphaving 1-3 heteroatoms (N, O or S) and a total of 5-14 ring atoms; andin addition two R₁₆ together with the nitrogen to which they areattached can form an unsubstituted heterocyclic or heteroaryl ringmoiety.

In specific embodiments M is a reactive group and more specifically anamine-reactive group. In specific embodiments, M is an amine-reactivegroup or a spacer moiety substituted with an amine-reactive group forforming one or more amide bonds to a cargo molecule comprising one ormore amine group. In specific embodiments, M is M1 which is a latentreactive group or a spacer moiety substituted with a latent reactivegroup, which latent reactive group does not react with any reactivegroup in the compound of formula IA or IB, or in any other M group inthe in compound, and which is selectively reactive, or can beselectively activated for reaction, after the compound is bonded to thecargo molecule. A latent reactive group can, for example, be activatedfor reaction inside of a cell for example by enzyme action inside of acell. A latent reactive group can for example be activated by action ofan esterase, for example after the cargo is delivered to a cell. Inspecific embodiments, M is M2 and is a spacer moiety substituted with areactive group. More specifically, M2 is a spacer moiety comprising alatent reactive group and substituted with a reactive group for forminga bond to the cargo molecule wherein the latent reactive group does notreact with the reactive group or the cargo molecule and can beselectively reacted or activated for reaction after the cargo moleculeis boronated. In specific embodiments, the reactive group of M2 is anamine-reactive group. In specific embodiments, the compound of formulaIA or IB comprises two -M groups, which more specifically are M1 whichis or which comprises a latent reactive group and M2 which is a spacermoiety which comprises a reactive group for bonding to cargo and alatent reactive group. In a specific embodiments, the latent reactivegroups of M1 and M2 cooperate to effect cleavage of the linker and tocleave the phenyl boronate groups from the cargo molecule.

In an additional embodiment, the invention provides a method forpreparation of boronated cargo, boronated peptides, and particularlyboronated oligopeptides, by solid phase peptide synthesis employingphenylboronated protected amino acids. Boronated cargo molecules, whichare not peptides or proteins, are prepared by ligation of a boronatedpeptide, particularly a boronated oligopeptide, to the cargo molecule.In a specific embodiment, boronated oligopeptides are prepared by Fmocsolid phase peptide synthesis employing Fmoc-protected boronated aminoacids. In a more specific embodiment, Fmoc-protected boronated glutamicacid or Fmoc-protected boronated phenyl alanine are employed to preparepeptides, including oligopeptides which contain one or more boronatedglutamic acid residues, one or more boronated aspartic acid residues,one or more boronated phenyl alanine residues or a combination of suchamino acid residues.

The invention also specifically provides various Fmoc-protectedphenylboronated amino acids for use in solid phase peptide synthesis.The invention also provides a method for boronating a peptide or proteinwhich comprises the step of binding or ligating or crosslinking aboronated oligopeptide reagent with the peptide or protein.

The invention further provides a method for enhancing cellular uptake ofa cargo molecule by boronating the peptide or protein with one or morephenylboronate groups and thereafter contacting the boronated peptide orprotein with a selected cell. In a specific embodiment, cargo moleculeis boronated with two or more phenylboronate or benzoboroxole groups. Ina specific embodiment, the phenylboronate is a compound of formula IA orIB. In specific embodiments, the phenylboronate is a benzoboroxole (alsocalled a benzoxaborole) compound of formula IB, wherein x is 1. Inspecific embodiments, the cargo molecule is boronated by binding,ligation or crosslinking of a boronated oligopeptide to a peptide orprotein. In specific embodiments, the cargo molecule is a peptide,protein or a nucleic acid.

More specifically, the invention provides a method for improved deliveryof a cargo molecule to a eukaryotic cell, particularly a mammalian cell,which comprises the step of contacting the cell or tissue containing thecell with a boronated cargo molecule. In a specific embodiment, the stepof contacting the cell or tissue is conducted in vitro. In a specificembodiment, the step of contacting the cell or tissue is conducted invivo. In a specific embodiment, the boronated cargo molecule comprises aboronated oligopeptide moiety of this invention. In specificembodiments, the cargo molecule carries two or more phenylboronategroups. In specific embodiments, the cargo molecule is a peptide orprotein which is boronated by reaction at one or more carboxylate groupsin the peptide or protein. In specific embodiments, the cargo moleculeis a peptide or protein which is boronated by reaction at one or moreamine groups in the peptide or protein. In a specific embodiment, thecargo protein is an enzyme. In a specific embodiment, the cargo proteinitself is not glycosylated (i.e., is not a glycoprotein). In a specificembodiment, the boronated cargo protein retains at least 10% of aselected biological activity of the protein prior to boronation. In aspecific embodiment, the boronated cargo enzyme retains at least 10% ofthe activity of the enzyme prior to boronation. In another specificembodiment, the cargo peptide or protein is an antibody or functionalfragment thereof and more specifically is a monoclonal antibody orfunctional fragment thereof.

In specific embodiments, the cargo molecule is a nucleic acid which isboronated by reaction at one or more amino groups in the nucleic acid.In a specific embodiment, the cargo protein is an enzyme. In a specificembodiment, the cargo protein itself is not glycosylated (i.e., is not aglycoprotein). In a specific embodiment, the boronated cargo nucleicacid retains at least 10% of a selected biological activity of thenucleic acid prior to boronation. In a specific embodiment, thebiological activity retained is binding of the nucleic acid tocomplementary nucleic acid.

The invention further provides kits for enhanced cellular uptake of apeptide or protein which comprise one or more of the phenylboronatecompounds of this invention or one or more of the boronated oligopeptidereagents of the invention which are individually packaged therein inselected amounts for use in boronating one or more peptides or proteinsfor enhanced uptake. The invention also provides kits for boronation ofa peptide or protein which comprise one or more of the phenylboronatecompounds of this invention or one or more of the boronated oligopeptidereagents of the invention which are individually packaged therein inselected amounts. Reagent kits may further comprise one or more solventsor reagents for carrying out binding, ligation, crosslinking or reactionof a phenylboronate or boronated oligopeptide with a selected peptide orprotein. Kits for enhanced cellular uptake may further comprise one ormore selected peptides or proteins to be delivered to cells, optionalreagents for labeling the peptide or protein, or reagents, media orsolvents for contacting cells with the boronated peptide or protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate, respectively, a scheme showing exemplaryboronation of RNase A with benzoboroxole groups (A) and its putativemechanism for expediting cellular delivery (B). The location of eachcarboxyl group of RNase A is depicted in the ribbon diagram (PDB entry7rsa; A. Wlodawer, L. A. Svensson, L. Sjölin, G. L. Gilliland,Biochemistry 1988, 27, 2705-271.)

FIG. 2 is an elution profile of a mixture of unmodified RNase A (elutingin region “A”) and boronated RNase A (eluting in region “B”) from acolumn of immobilized heparin in the absence (solid line) or presence(dashed line) of fructose (0.10 M).

FIG. 3 shows the results of fluorescence polarization assay ofribonucleases binding ganglioside-labeled liposomes in the presence orabsence of 10 mM fructose. Data was normalized to polarization of eachribonuclease incubated with non-extruded DOPC lipids. Data pointsrepresent the mean (±SD) of triplicate experiments. Asterisks indicatevalues with p<0.05.

FIG. 4A is a graph of the results of flow cytometry experimentsmeasuring internalization of unmodified and benzoboroxole-boronatedRNase A into Pro-5 and Lec-2 cells in the absence or presence offructose (0.25 M). Flow cytometry data were normalized to theinternalization of unmodified RNase A into Pro-5 cells. Error barsrepresent the SD.

FIG. 4B is a confocal microscopy image of live Pro-5 cells incubated for4 h with benzoboroxole-boronated RNase A (5 μM) that had been labeledcovalently with a green fluorophore. Nuclei were stained blue withHoechst 33322 (2 μg/mL). Scale bar: 10 μm.

FIG. 4C is a graph of the results of flow cytometry experimentsmeasuring internalization of unmodified and phenylboronate-boronatedRNase A into Pro-5 and Lec-2 cells in the absence or presence offructose (0.25 M). Flow cytometry data were normalized to theinternalization of unmodified RNase A into Pro-5 cells. Error barsrepresent the SD.

FIG. 5 is a graph showing inhibition of K-562 cell proliferation byunmodified and boronated RNase A. (, closed circles) Unmodified RNase A(IC50>50 μM); (♦, closed diamonds) boronated RNase A (IC50 4.0±0.3 μM);(⋄, open diamonds) boronated RNase A in the presence of fructose (50 mM)(IC50 9±1 μM); (□, open squares) boronated RNase A alkylated with2-bromoacetate (IC50>50 μM). The proliferation of K-562 cells wasmeasured by the incorporation of [methyl-3H]thymidine. Data pointsrepresent the mean (±SEM) of three separate experiments performed intriplicate.

FIG. 6 is a graph of the results of flow cytometry experiments measuringinternalization of unmodified lysozyme compared to lysozyme boronatedwith benzoboroxole into HeLa cells in the presence and absence offructose. Error bars represent the SD.

FIG. 7 is a graph of the results of flow cytometry experiments measuringinternalization into HeLa cells of unmodified avidin compared to avidincomplexed to PBA-boronated biotin peptide in the presence and absence offructose. Error bars represent the SD.

FIGS. 8-12 illustrate exemplary reactive groups and exemplary reactionsthat can be employed to boronate peptides or proteins with phenylboronicacids. 2-substituted phenylboronic acid is used in these figures toillustrate exemplary reactions. The exemplified reactive groups can besubstituted on other ring positions of the phenylboronic acid and otherring positions may be further substituted as described herein.Additionally, analogously substituted phenylboroxoles can be used toboronate peptides and proteins. Examples are given in which the reactivegroup is directly bonded to the ring of the phenylboronic acid and inwhich the reactive group is indirectly attached to the phenylboronicacid via a spacer moiety. It will be appreciated by one of ordinaryskill in the art that a variety of art-known linking moieties can beemployed which are suitable for boronation of peptides and proteinsusing the illustrated reactions.

FIG. 13 illustrates the determination of the ¹H NMR peaks correspondingto the aryl protons in bound and free boronic acid. (A) ¹H NMR spectrumof solution 1. (B) ¹H NMR spectrum of solution A. (C) Overlay ofaromatic region of spectra from panels A and B. (D) Example of aspectrum that was interpreted using the overlay from panel C, and usedto determine the value of K_(a) for fructose with PBA. The [B·S]/[B]ratio was calculated from the isolated peaks for the complex (3H,7.01-7.11 ppm) and the isolated peaks for the free boronic acid (2H,7.54-7.58 ppm).

FIG. 14 is a representative ¹H NMR spectrum that was used to determinethe K_(a) value for benzoboroxole and fructose (10.3 mM). Peakscorresponding to the aryl protons in bound and free boronic acid weredetermined as described in Example 2 and FIG. 13. The [B·S]/[B] ratiowas calculated from the isolated peaks for the complex (¹H, 7.31-7.38ppm, a mixture of isomeric species) and the isolated peaks for the freeboronic acid (1H, 7.43-7.53 ppm, a mixture of isomeric species).Additional saccharide decreased the integration of the small peak at7.44 ppm equally with that at 7.49 ppm, which arise from free boronicacid; and the shoulder peak at 7.31 ppm increased equally with that at7.35 ppm, which arises from the complex.

FIG. 15 is a representative ¹H NMR spectrum that was used to determinethe K_(a) value for PBA and glucose (44.9 mM). Peaks corresponding tothe aryl protons in bound and free boronic acid were determined adescribed above. The [B·S]/[B] ratio was calculated from the isolatedpeaks for the complex (3H, 7.09-7.21 ppm) and the peaks for the freeboronic acid (2H, 7.64-7.68 ppm).

FIG. 16 is a representative ¹H NMR spectrum that was used to determinethe K_(a) value for benzoboroxazole and glucose (32.9 mM). Peakscorresponding to the aryl protons in bound and free boronic acid weredetermined a described above. The [B·S]/[B] ratio was calculated fromthe isolated peaks for the complex (¹H, 7.29-7.34 ppm) and the isolatedpeaks for the free boronic acid (¹H, 7.45-7.51 ppm). Note the broadeningof the aryl protons, which had been reported for NMR spectra of boronicacids in the presence of pyranose sugars. (M. Bérubé, M. Dowlut, D. G.Hall, J. Org. Chem. 2008, 73, 6471-6479)

FIG. 17 is a representative ¹H spectrum that was used to determine theK_(a) value for PBA with Neu5Ac (35.4 mM). Peaks corresponding to thearyl protons in bound and free boronic acid were determined a describedabove. The [B·S]/[B] ratio was calculated from the isolated peaks forthe complex (3H, 7.15-7.26 ppm) and the isolated peaks for the freeboronic acid (2H, 7.6-7.69 ppm). Note that the aryl peaks have beenbroadened by the addition of the saccharide.

FIG. 18 illustrates the ¹H NMR spectrum of Neu5Ac. This spectrumindicates a small peak that overlapped with the aromatic regions of theboronic acids. This peak was subtracted out of all NMR spectra used toevaluate the interaction of benzoboroxazole and Neu5Ac.

FIG. 19 is a representative ¹H spectrum that was used to determine theK_(a) value for benzoboroxazole with Neu5Ac (14.4 mM). Peakscorresponding to the aryl protons in bound and free boronic acid weredetermined as described in Example 2. The [B·S]/[B] ratio was determinedfrom the isolated peaks for the free boronic acid (1H, 7.44-7.51 ppm)and the remainder of the aromatic region (7.15-7.36 ppm), whichrepresented 3H from the free boronic acid and all 4 aromatic protonsfrom the complex. Unlike fructose and glucose, the single isolatedproton of the complexed species (7.33 ppm) was too broad to integrateaccurately, and the entire region was used instead.

FIG. 20 illustrates MALDI-TOF spectra of (A) unmodified RNase A and (B)boronated RNase. Data were fitted to a Gaussian curve (red line). Theobserved molecular mass of unmodified RNase A (13,659 Da) was subtractedfrom the observed molecular mass of boronated RNase A (14,641 Da) togive 982 Da. This value was divided by the molecular mass of5-amino-2-hydroxymethylphenylboronic acid after correcting for the waterlost during conjugation (148.95 Da−18.02 Da=130.93 Da) to give 7.5±2.0boronic acids conjugated to RNase A, where SD=2.0 arises from the SD ofthe Gaussian fit, 265.2 Da, divided by 130.93 Da.

FIG. 21 illustrates fluorescence polarization data for the binding ofphenylboronate-conjugated RNase A to GD3 ganglioside in liposomes.BODIPY FL-labeled phenylboronate-conjugated RNase A was incubated withliposomes containing GD3 ganglioside in 25 mM HEPES buffer, pH 7.0,containing NaCl (75 mM). Data points represent the mean (±SE) ofduplicate experiments. Data were fitted to a binding isotherm asdescribed in M. H. Roehrl, J. Y. Wang, G. Wagner, Biochemistry 2004, 43,16056-16066 to give Kd=(54±11) μM.

FIG. 22 illustrates a scheme for cellular uptake mediated by boronylpendants. Boronate 10 is an exemplary boronation reagent (an exemplarybenzoboroxole) of the invention used to acylate the amino groups of acargo molecule, such as a target protein or nucleic acid. Multivalentcomplexation with cell-surface glycans facilitates entry into endosomes,where esterases will release intact protein. The fluorescence of thecoumarin product can be employed to determine uptake.

FIG. 23 illustrates synthesis of exemplary boronation reagents of theinvention. Synthesis of the phenylboronate 20 is illustrated. Theexemplary benzoboroxole is prepared by an analogous synthesis whereinthe —CH₂—O—B(OH)— moiety of the benzoboroxole is installed by methodsthat are well known in the art.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based at least in part on the demonstration thatpendant phenylboronic acids mediate the delivery of molecules (cargo)into mammalian cells. More specifically, bonding of one or morephenylboronic acids to such molecules, particularly nucleic acids, andpeptides or proteins generally enhances uptake of the boronated cargomolecule into mammalian cells. Additionally, in specific embodiments,the boronated cargo molecule retains biological activity of thecorresponding non-boronated cargo molecule. FIG. 22 illustrates use of aboronation reagent of the invention (compound 10) to boronate a proteinand further illustrates the scheme for cellular uptake of cargo(exemplified by protein). Multivalent complexation with cell-surfaceglycans is believed to facilitate entry into endosomes, where cargo isreleased, for example by enzyme action. In the illustrated scheme,esterases release intact cargo protein. In the illustrated example,fluorescence of the coumarin by-product of release can be employed toassess uptake of cargo molecule.

More specifically herein, boronation of peptides and proteins, e.g.,RNase A, lysozyme and avidin, is shown to generally enhance uptake ofthe boronated peptide or protein into mammalian cells.

The term “enhancement of cellular uptake” refers to enhancement ofuptake of a boronated cargo molecule compared to uptake of the analogousnon-boronated cargo molecule. Enhancement of cellular uptake can bemeasured by any art-known method and useful methods are exemplified inthe examples. In specific embodiments, enhancement of cellular uptake of2-fold or higher relative to the non-phenylboronated cargo molecule isobtained. Enhancement of cellular uptake may be assessed in terms of %internalization compared to non-boronated cargo molecule. In specificembodiments, enhancement of % internalization of 50% or more compared tocontrols is obtained.

Enhanced delivery of boronated cargo, particularly boronated proteins,e.g., boronated RNase A is described hereafter in more detail.

In specific embodiments, the invention provides methods and reagents forboronating cargo molecules, and particularly boronating of amino acids,peptides, proteins, nucleic acids and analogs thereof and nucleosidesand analogs thereof with phenylboronate or benzoboroxole groups toenhance uptake of such cargo molecules into cells.

In specific embodiments, the invention provides methods and reagents forboronating nucleotides, nucleic acids and analogs thereof.

In specific embodiments, the invention provides methods and reagents forboronating peptides and proteins.

In specific embodiments, the invention provides methods and reagents forboronating cargo molecules which contain one or more amino groupswherein boronation is facilitated by formation of one or more amidebonds.

Herein, phenylboronate groups are chemical groups in which a

is directly attached to an optionally substituted phenyl ring, where Ris an optionally substituted aliphatic group which optionally links tothe phenyl ring to form a 5- or 6-member boroxole ring. In specificembodiments, the boronate group is:

where R₇, R₈ and x are defined above. In specific embodiments, thephenyl ring of the phenylboronate is also substituted with one, two,three or four non-hydrogen substituents. In specific embodiments, thephenyl ring of the phenylboronate is also substituted with one, two,three or four non-hydrogen substituents which are electron withdrawinggroups. Substitution of the phenyl ring with one or more electronwithdrawing groups is believed to enhance binding of the boronate groupto diols, particularly diols of saccharides. In specific embodiments,phenylboronates of this invention are not bound to a solid, such as aresin or other polymeric material and are not bound to a surface.Phenylboronates useful in the present invention include those offormulas IA and IB and salts thereof as described above.

In specific embodiments of formula IB, x is 1 and the boronating speciesis a benzoboroxole. In specific embodiments, each R₇ and R₈ isindependently hydrogen or a methyl group. In specific embodiments, thecompound of formula IA or IB contains a single -M group. In specificembodiments, the compound of formula IA or IB contains a single -M groupand one of R₂-R₄ is -M. In specific embodiments, R₂ or R₄ is -M.

In specific embodiments, the compound of formula IA or IB contains two-M groups, one of which is M1 and the other of which is M2, where M1 isa latent reactive group and M2 is a spacer substituted with a reactivegroup and which contains a latent reactive group. More specifically, thelatent reactive groups of M1 and M2 function together to cleave theboronate from the cargo molecule on selective activation of one or bothlatent reactive groups.

In specific embodiments of formula IA and IB, M1 and M2 are substitutedon adjacent ring carbons in the phenyl ring. More specifically, R₂ is M1and R₃ is M2; R₂ is M2 and R₃ is M1; R₄ is M1 and R₃ is M2; R₃ is M1 andR₄ is M2; R₄ is M1 and R₅ is M2; R₄ is M2 and R₅ is M1; R₁ is M1 and R₂is M2; R₁ is M2 and R₂ is M1; R₄ is M1 and R₆ is M2; or R₄ is M2 and R₆is M. More specifically in compounds of formula IA, R₂ is M1 and R₃ isM2; R₂ is M2 and R₃ is M1; R₄ is M1 and R₃ is M2; or R₃ is M1 and R₄ isM2 and more specifically in compounds of formula IB R₄ is M1 and R₃ isM2; or R₃ is M1 and R₄ is M2.

In specific embodiments of formula IA or IB, R₁, R₅ and R₆ areindependently selected from hydrogen, an optionally substitutedstraight-chain or branched aliphatic group having 1-8 carbon atoms, a—CO₂R₁₀ group, a —O—CO—R₁₀ group, a —CON(R₁₂)₂ group, a —O—CO—N(R₁₂)₂group, a —N(R₁₂)₂ group, a —OR₁₀ group, a —(CH₂)_(m)—OR₁₀ group, a—(CH₂)_(m)—N(R₁₂)₂ group, a halogen, a nitro group, or a cyano group,where variables are as defined above. In more specific embodiments, R₁and R₅ or R₆ are independently selected from hydrogen; C1-C3 alkylgroup; —CO₂H; —CONH₂; —NH₂; —CON(R₁₂)₂ or —N(R₁₂)₂, where R₁₂ is a C1-C6alkyl group; a hydroxyl; a C1-C3 alkoxyl; a —(CH₂)_(m)—OH or—(CH₂)_(m)—NH₂ group where m is 1-3; halogen, nitro group or cyanogroup.

In specific embodiments of formula IA or IB, R₂, R₃ and R₄ areindependently selected from hydrogen, -M, an optionally substitutedstraight-chain or branched aliphatic group having 1-8 carbon atoms, a—CO₂R₁₀ group, a —O—CO—R₁₀ group, a —CON(R₁₂)₂ group, a —O—CO—N(R₁₂)₂group, a —N(R₁₂)₂ group, a —OR₁₀ group, a —(CH₂)_(m)—OR₁₀ group, a—(CH₂)_(m)—N(R₁₂)₂ group, a halogen, a nitro group, or a cyano group,where variables are as defined above, wherein at least one of R₂-R₄ is-M. Additionally, two adjacent R₂-R₆, together with the ring carbons towhich they are attached, optionally form a 5- or 6-member alicyclic,heterocyclic, aryl or heteroaryl ring moiety wherein the ring membersare optionally substituted.

In specific embodiments, variable groups of formulas IA and IB areoptionally substituted as defined above. In more specific embodiments,optional substitution of compounds of formulas IA or IB is substitutionof a group or moiety by 1-5 substituents selected from substituents asnoted above. In additional embodiments, optional substitution ofcompounds of formulas IA or IB is substitution of a group or moiety by 1or 2 substituents selected from substituents as noted above. In specificembodiments, optional substituent groups have 10 or less carbon atoms.In specific embodiments, optional substituent groups have 12 or lessatoms. In specific embodiments, optional substituent groups have 6 orless carbon atoms. In specific embodiments, groups as defined forformulas IA and IB are unsubstituted. In specific embodiments,optionally substitution is substitution by one to 7 substituentsselected from halogen (F, Cl, Br or I); nitro group; cyano group; aC1-C3 alkyl group; a C1-C3 alkoxy group; a C2-C3 alkenyl group; a C2-C3alkynyl group; a 5- or 6-member alicyclic ring group, wherein one or tworing carbons are optionally replaced with —CO— and which may contain oneor two double bonds; phenyl group; benzyl group; naphthyl group;biphenyl group; —COH; —CO₂H; —CONH₂; —NH₂; —OH; —SH or —SO₃H, whereinalkyl, alkenyl, alkynyl, alicyclic ring, phenyl; benzyl; naphthyl, andbiphenyl groups are optionally substituted with one or more halogen, —OHgroup, —SH group, methyl group, methoxy group, trihalomethyl group,cyano group, or nitro group.

In specific embodiments herein, R₁-R₆ that are not -M do not contain areactive group as described herein that is in the -M group. In specificembodiments herein, R₁-R₆ that are not -M may contain reactive groups asdefined herein if those reactive groups are protected with anappropriate protective group which selectively prevents their reaction.Such protective groups may be removed if desired after boronation of thepeptide or protein.

In specific embodiments, -M is a reactive group or a spacer moietycarrying a reactive group wherein the reactive group:

(1) reacts with one or more of: an amine group, a carboxylic acid group,a sulfhydryl group or a hydroxyl group, particularly where that group isa group of a natural or unnatural amino acid of such an amino acid of apeptide or a protein;(2) reacts with an aldehyde or ketone group, an azide group, anactivated ester group, a thioester group, phosphinothioester, or othergroup which is introduced into or generated in the amino acid, peptideor protein; or(3) reacts with one reactive group of a homobidfunctional or aheterobifunctional crosslinking reagent,to bond or attach the phenylboronate group substituted with the M groupto an amino acid, peptide or protein.

In another embodiment, M is or contains a reactive group that can beligated to a peptide or protein by a peptide ligation method. Inspecific embodiments, M is or contains an amine group, a carboxyl groupor ester thereof, an activated ester group, an azide, a thioester, or aphosphinolthioester.

In general the optional spacer moiety of the M group is compatible withthe reactive group therein (e.g., does not detrimentally affectreactivity of the reactive group) and the spacer itself is not reactivewith the compounds to be conjugated. In specific embodiments, the spacermoiety contains from 3-20 atoms (typically C, O, S and/or N atoms whichmay be substituted with H or non-hydrogen substituents), includingresidues from the reactive group), and optionally contains one or morecarbon-carbon double bonds, and/or a 5- to 8-member alicyclic, a 5- to8-member heterocyclic, a 6- or 10-member aryl or a 5- or 6-memberheteroaryl ring. Carbon atoms in the spacer or linker are optionallysubstituted with one or more hydroxyl groups, oxo moieties (═O), orhalogens (e.g., F). Nitrogen groups in the spacer may be substitutedhydrogen or with C1-C3 alkyl groups. The spacer may contain one or two—S—S— and/or —SO₂— moieties. The spacer may contain a diol(>C(OH)—C(OH)<) moiety.

In a specific embodiment, the spacer in the linker comprises one or moreether linkages. More specifically, the spacer is or comprises a—O—CH₂—O— moiety. In a specific embodiment, the linker between thephenyl boronate and the cargo molecule is or comprises an acetoxymethylether. More specifically, the linker is or comprises a —O—CH₂—O—CO—moiety. In a specific embodiment of formula IA or IB, any of R₁-R₆ is—O—CH₂—O—CO—CH₃ or comprises a —O—CH₂—O— or a —O—CH₂—O—CO— moiety. In aspecific embodiment, M comprises a —O—CH₂—O— or a —O—CH₂—O—CO—. See:Lavis et al. (2011) Chemical Science 2:521-530.

The spacer may be selectively cleavable, for example, by change ofconditions (e.g., pH change), addition of a cleavage reagent, orphotoirradiation (e.g., UV irradiation). Such selectively cleavablespacers contain a latent reactive group which is selectively reactive orcan be selectively activated for reaction. In specific embodiments, acleavable spacer includes a disulfide bond which is selectivelycleavable, for example on treatment with dithiothreitol, a diol moietywhich is selectively cleavable by treatment for example with periodate,an ester moiety, which is selectively cleavable by treatment withhydroxylamine, or a sulfone moiety (—SO₂—) which is selectivelycleavable under alkaline conditions.

In specific embodiments, -M is selected from: —X of -LX, where X is thereactive group for ligation, bonding or crosslinking to an amino acid,peptide or protein and L is a spacer moiety. A variety of spacermoieties are known in the art to be useful for bioconjugation ofmolecules to amino acids, peptides and proteins. All such art-knownspacer moieties can be employed in this invention, if compatible withthe chemistry of the phenylboronate and the amino acid, peptide orprotein and which do not detrimentally affect reactivity of chosenreactive groups and which do not themselves react with thephenylboronate, amino acid, peptide, protein or reactive groups.

In specific embodiments, L is selected from the following divalentmoieties:

—Y1-[1-Y3-, where Y1 and Y3 are optional and may be the same ordifferent;—Y1-L1-L2-Y3-, where Y1 and Y3 are optional and may be the same ordifferent and L1 and L2 are different; or—Y1-[1-[L2-Y2]_(y)-L3-Y3-, where Y1 and Y3 are optional, Y1, Y2 and Y3may be the same or different, L1 and L3 are optional and L1, L2 and L3may be the same or different and y is an integer indicating the numberof repeats of the indicated moiety;

wherein each L1-L3 is independently selected from an optionallysubstituted divalent aliphatic, alicyclic, heterocyclic, aryl, orheteroaryl moiety having 1 to 30 atoms and each Y1, Y2 and Y3 isindependently selected from: —O—, —S—, —NRc-, —CO—, —O—CO—, —CO—O—,—CO—NRc-, —NRc-CO—, —NRc-CO—NRc-, —OCO—NRc-, —NRc-CO—O—, —N═N—,—N═N—NRc-, —CO—S—, —S—CO—, —S—S—, —SO₂—, —CRc(OH)—CRc(OH)—, where Rc ishydrogen or C1-C3 alkyl.

In specific embodiments, y is 1-12 and L1-L3 are selected from: —(CH₂)y-(an alkylene) wherein one or more, and preferably 1-4, carbons of thealkylene are optionally substituted with one or more non-hydrogensubstituents selected from halogens, C1-C3 alkyl groups or hydroxylgroups, preferred y are 2-6;

a cycloalkylene, having a 3-8-member ring wherein one or more, andpreferably 1-4, carbons of the cycloalkylene are optionally substitutedwith one or more non-hydrogen substituents selected from halogens, C1-C3alkyl groups or hydroxyl groups, including among others a1,4-cyclohexylene, a 1,3-cylohexylene, a 1,2-cyclohexylene; a1,3-cyclopentylene, each of which is optionally substituted;

a phenylene, wherein 1-4 of the ring carbons are optionally substitutedwith one or more non-hydrogen substituents selected from halogens, C1-C3alkyl groups, nitro group, cyano group, or hydroxyl groups, including a1,4-phenylene, a 1,3-phenylene or a 1,2-phenylene, each of which isoptionally substituted;

a naphthylene, wherein 1-8 of the ring carbons are optionallysubstituted with one or more non-hydrogen substituents selected fromhalogens, C1-C3 alkyl groups, nitro group, cyano group, or hydroxylgroups, including a 2,6-naphthylene, a 2,7-naphthylene, a1,5-naphthylene, or a 1,4-naphthylene moiety, each of which isoptionally substituted;

a biphenylene, wherein 1-8 of the ring carbons are optionallysubstituted with one or more non-hydrogen substituents selected fromhalogens, C1-C3 alkyl groups or hydroxyl groups, including a1,4′-biphenylene, a 1,3′-biphenylene or a 1,2′-biphenylene, each ofwhich is optionally substituted;

an alkenylene, i.e., a divalent alkylene group, containing one or more,preferably 1 or 2 double bonds and having 2-12 and preferably 2-8 carbonatoms, wherein one or more, and preferably 1-4, carbons are optionallysubstituted with one or more non-hydrogen substituents selected fromhalogens, C1-C3 alkyl groups or hydroxyl groups, including among others,—CH═CH— and —CH═CH—CH═CH— which are optionally substituted;

a heterocyclene (i.e., a divalent heterocyclic moiety) having a3-8-member ring with 1-3 heteroatoms, selected from N, O or S, whereinone or more, and preferably 1-4 carbons, or where feasible heteroatoms,of the heterocyclene are optionally substituted with one or morenon-hydrogen substituents selected from halogens, C1-C3 alkyl groups,nitro groups, or hydroxyl groups, including among others a2,4-3H-azepinylene moiety, a piperidinylene (e.g., a1,4-piperidinylene), a piperazinylene (e.g., a 1,4-piperazinylene), atriazolidinylene (a divalent triazolidinyl) or a triazolylene (adivalent triazolyl) each of which is optionally substituted; or

a heteroarylene (i.e., a divalent heteroaryl moiety) having a 5- or6-member heteroaryl ring having 1-3 heteroatoms selected from N, O or S,wherein one or more, and preferably 1-2 carbons, or where feasibleheteroatoms, of the heteroarylene are optionally substituted with one ormore non-hydrogen substituents selected from halogens, C1-C3 alkylgroups, nitro groups, or hydroxyl groups, including among others apyridylene (e.g., 2-5-pyridylene), imidazolylene (e.g.,2,5-imidazolylene, 4,5-imidazolylene), each of which is optionallysubstituted.

In additional embodiments, the spacer is an ethylene glycol spacer. Morespecifically, -M is selected from —[(CH₂)_(y)—O]_(a)—, where y is 1-4and a is 1-6, and preferably 1-3.

In additional embodiments, -M is selected from:

—CO—NH—CRaRb-[CO—NH—CRaRb]_(a)—CO—OH, where a is 1-6;—COO—CRaRb-[CO—NH—CRaRb]_(a)—CO—OH, where a is 1-6;—O—CO—NH—CRaRb-[NH—CO—CRaRb]_(a)—NH₂, where a is 1-6;—Y4-CRaRb-[W—CRaRb]_(a)—X4, where W is —NH—CO— or —CO—NH—, where a is1-6;where:

—X4 is a functional group that reacts with one or more of an aminegroup, a carboxylic acid group or ester thereof, a sulfhydryl group, ahydroxyl group, an azide group, a thioester group, a phoshinothioestergroup, an aldehyde group or a ketone group of an amino acid, peptide orprotein; and

—Y4- is —O—, —S—, —NH—, —CO—, —CO₂—, —O—CO—, —CO—O—, —CO—NRc-, —NRcCO—,—CO—S—, or —S—CO— and Rc is hydrogen or a C1-C3 alkyl;

Ra and Rb are selected independently from hydrogen, a C1-C8 aliphaticgroup, an alicylic, a heterocyclic, an aryl or a heteroaryl group, eachof which is optionally substituted or

Ra is hydrogen and Rb is a side-group or protected side-group of aproteinogenic amino acids or an amino acid selected from hydroxyproline,ornithine, or citrulline.

In specific embodiments, the phenylboronic acid, except for the -M groupor any salt counterion thereof, contains at most 20 carbon atoms.

In specific embodiments, X and X4 are —NH₂, —COOH or an activated esterthereof, —SH, —N₃, —COH, —CO—CH═CH₂, —NH—CO—CH═CH, or —C≡CH

In specific embodiments, the invention provides boronation reagents offormulas IA and IB wherein M1 is a latent reactive group which can beactivated to form —O⁻, for example by cleavage of an ester bond (e.g.,—O—CO—R₂₄, where R24 is an optionally substituted alkyl or aryl groupand specifically can be a —OCO—CH₃ group, cleavage of a —O—PO₃H₂ orrelated phosphate ester or cleavage of a —O—SO₂—R₂₅ group where R₂₅ isan substituted alkyl or aryl group, particularly a halogenated alkyl ora phenyl substituted with one or more halogens, nitro groups or otherelectron withdrawing groups. For example, M1 can be activated by anesterase, an alkaline phosphatase or an exogenous thiol. In thisembodiment, M2 carries a latent reactive group which cooperates with the—O— generated to cleave the M2 linker and cleave the boronate from thecargo molecule. IN specific embodiments, M2 carries a trimethyl lockstructure or a coumarin-based structure. See: Levine and Raines (2012)Chemical Science 3(8):2412-2420; Zheng, A.; Wang, W.; Zhang, H.; Wang,B. Tetrahedron 1999, 55, 4237-4254; Liao, Y.; Wang, B. Bioorg. Med.Chem. Lett. 1999, 9, 1795-1800; Gomes, P.; Vale, N.; Moreira, R.Molecules 2007, 12, 2484-2506; Simplicio, A. L.; Clancy, J. M.; Gilmer,J. F. Molecules 2008, 13, 519-547; and Wang, W.; Camenisch, G.; Sane, D.C.; Zhang, H.; Hugger, E.; Wheeler, G. L.; Borchardt, R. T.; Wang, B. J.Control. Release 2000, 65, 245-251. Each of these references isincorporated herein for details on Specific M1 and M2 groups useful inthis invention.

In specific embodiments, M2 is:

where R₃₀ and R₃₁ are H, alkyl (particularly C1-C3 alkyl) or R₃₀ and R₃₁together with the intervening C═C moiety form a 6-8 member carbocyclicor heterocyclic ring wherein one of the ring carbons can be replacedwith —CO—, such as a cyclohexenyl ring or cyclohexenone ring and —COX isan activated ester (e.g., X is a good leaving group), such as NHS esters(N-hydroxysuccinimide esters) or sulfo NHS esters(N-hydroxysulfosuccinimide esters).In specific embodiments, M2 is:

where —COX is an activated ester. More specifically, in compounds offormula IA and IB containing this M2, it is preferred that an adjacentcarbon on the phenyl ring carries a methyl or a C2-4 alkyl group.

The invention further provides boronation reagents of the followingformulas:

or salts thereof,

where R₁-R₄ take values as defined for formulas IA and IB, except thatin the reagent of formula IIA one of R₂ or R₃ is —B(OH)₂ or R₂ and R₃together are:

where R₇ and R₈ are defined above and preferably both are H and x is 1or 2 and preferably 1;R₃₀ and R₃₁ are as defined above;X—CO— is an activated ester and—O—Y is a precursor to —O⁻, i.e., Y is a labile group, as describedabove, and more specifically —OY is —O—CO—R₂₄, where R²⁴ is anoptionally substituted alkyl or aryl group, a —O—PO₃H₂ or correspondingphosphate ester or a —O—SO₂—R₂₅ group, where R₂₅ is an substituted alkylor aryl group, particularly a halogenated alkyl or an aryl groupsubstituted with one or more halogens, nitro groups or other electronwithdrawing groups. In a specific embodiment, R₂₅ is a phenyl groupsubstituted with one or more halogens, nitro groups or other electronwithdrawing groups.

The invention also provides boronation reagents of the followingformulas:

or salts thereofwhere R₁-R₄ take values as defined for formulas IA and IB, except thatin the reagent of formula IVA one of R₂ or R₃ is —B(OH)₂ or R₂ and R₃together are:

where R₇ and R₈ are defined above and preferably both are H and x is 1or 2 and preferably 1 and where in a specific embodiment R₄ is a methylgroup; X—CO— is an activated ester and—O—Y is a precursor to −0, i.e., Y is a labile group, as describedabove, and more specifically —OY is —O—CO—R₂₄, where R²⁴ is anoptionally substituted alkyl or aryl group, a —O—PO₃H₂ or correspondingphosphate ester or a —O—SO₂—R₂₅ group, where R₂₅ is an substituted alkylor aryl group, particularly a halogenated alkyl or an aryl groupsubstituted with one or more halogens, nitro groups or other electronwithdrawing groups. In a specific embodiment, R₂₅ is a phenyl groupsubstituted with one or more halogens, nitro groups or other electronwithdrawing groups.

The invention provides the following additional boronation reagents:

or salts thereof where X—CO—, Y—O—, R₃₀, R₃₁ are as defined above andRx, Ry and Rz take values of R₁-R₆ above with the exception that one ofthe Rz's is a phenylboronate group of formula IA or IB, wherein one ofR₁-R₆ provides a direct bond to or a linker to the phenyl ring of VI orVII. In a specific embodiment, the direct bond to the phenyl ring is aC—C bond, or a —O—C bond. In a specific embodiment, the linker is alinker as defined above or is selected from —(CH₂)_(a)—, —O—(CH₂)_(a)—,—S—(CH₂)_(a)—, —O—(CH₂)_(a)—O—, —(CH₂)_(a)—O—, —S—(CH₂)_(a)—S—,—CO—(CH₂)_(a)—, —OC—(CH₂)_(a)—, —CO—(CH₂)_(a)—CO—, —(CH₂)_(a)—CO—,—NHCO—(CH₂)_(a)—, —(CH₂)_(a)—CONH—, —NHCO—(CH₂)_(a)—CO— or—CO—(CH₂)_(a)—NHCO—; where a is an integer from 1-10 or 1-6.

Phenylboronate compounds of the invention can be prepared in view of thedescriptions herein and methods that are known in the art or by routineadaptation of such methods. Methods useful for synthesis ofphenylboronates of this invention can be found, for example, in U.S.Pat. Nos. 5,594,111; 5,594,151; 5,623,055; 5,777,148; 5,744,627;5,837,878; and 6,156,884, in references [27] and [29-36]. Each of thesereferences is incorporated by reference herein in its entirety fordescriptions of such useful synthetic methods. FIG. 23 illustratessynthesis of an exemplary boronation agent of the invention (compound20). The methods illustrated in FIG. 23 can be routinely adapted forsynthesis of benzoboroxoles such as compound 10 illustrated in FIG. 24.

In a related embodiment, the invention provides a phenylboronatedoligopeptide for ligation to, binding to or crosslinking with a cargomolecule, such as a nucleic acid, peptide or protein and the boronatedpeptide or protein which is ligated, bound to or crosslinked with thephenylboronated oligopeptide. In specific embodiments, thephenylboronated oligopeptide contains 2-30 amino acids, contains 2-aminoacids, contains 2-15 amino acids or contains 5-10 amino acids. Inspecific embodiments, 75% or fewer of the amino acids of theoligopeptide are boronated. In additional embodiments, 50% or fewer, 25%or fewer or 10% or fewer of the amino acids of the oligopeptide areboronated. In specific embodiments, a phenylboronate group is bonded toan amino acid side group of one or more amino acids of the oligopeptide.In a specific embodiment, a phenylboronate group is bonded to theN-terminus of the oligopeptide. In a specific embodiment, a phenylboronate is bonded to the C-terminus of the oligopeptide.Phenylboronated oligopeptides include those in which one or morephenylboronated amino acid, such as a boronated glutamate, aspartate,phenyl alanine or lysine alternate in the oligopeptide with one or morenon-boronated amino acid, such as glycine or leucine.

Phenylboronated oligopeptides include those containing one or morephenylboronated amino acids, such as a boronated glutamate, aspartate,phenyl alanine or lysine, wherein each boronated amino acid is spacedfrom other boronated amino acids by one or more non-boronated aminoacids, such as a glycine. In such embodiments, the phenylboronate groupsmay extend over the length of the oligopeptide. In other embodiments,the phenylboronate groups may be located on adjacent amino acids at oneend of the oligopeptide, for example at or near the end distal to thesite of attachment of the oligopeptide to the peptide or protein.

Any amino acid that is known in the art to be useful in the synthesis ofoligopeptides may be employed in the oligopeptides of this invention.Amino acids include any naturally-occurring amino acids or anynon-naturally occurring (synthetic) amino acids, and any amino acidsoccurring in peptides or proteins in nature.

In a specific embodiment, the invention provides a phenylboronatedoligopeptide reagent containing a ligand group, such as biotin or afunctional derivative thereof. The reagent generically has the formula:T-L-phenylboronated oligopeptide (where T-L may be at the N-terminus orC-terminus of the oligopeptide).

where T is a ligand group that binds to a cargo molecule, such as anucleic acid, peptide or protein or a reactive group X (as definedabove) that reacts with a reactive group of a cargo molecule, such as anucleic acid, peptide or protein, particularly that is anamine-reactive, a carboxyl-reactive, a sulfhydryl-reactive, ahydroxyl-reactive, an aldehyde- or ketone-reactive group, anazide-reactive, a thioester reactive or a phosphinothiol reactive groupand L is an optionally spacer moiety (as defined above).

On binding to or reacting with a cargo molecule, such as a nucleic acid,peptide or protein, the cargo molecule carries one or more boronatedoligopeptide tags: Protein (-T1-L-boronated oligopeptide)_(z), where zis 1-12 or more and preferably z is 1-6.

In a specific embodiment, the oligopeptide or oligopeptide reagentcomprises 2-20 amino acids of which at least two amino acids areboronated as described herein. In more specific embodiments, theoligopeptide carries 3-12 phenylboronate groups. In a specificembodiment, the peptide contains 1-12 amino acids with side groupshaving an —NH₂ group. In a specific embodiment, the peptide contains1-12 amino acids with side groups having an —COOH group.

In a specific embodiment, T is a ligand which binds to a selectednucleic acid or protein. In a specific embodiment T is biotin or aderivative thereof. Various functional biotin derivatives are known inthe art and a number are commercially available. For example, biotin canbe readily transformed at its carboxyl terminus for attachment tonumerous species. Further, a variety of biotinylation reagents are knownin the art which can be employed in preparation of boronatedoligopeptide reagents of this invention.

In a specific embodiment, T is a group that can function for peptideligation to form a peptide bond between the peptide or protein and theboronated oligopeptide. This group may be selected from —NH₂, —COOH oran activated ester, a thioester, an azide, a phosphinothioester or thelike.

In specific embodiments, spacer groups for phenylboronate compounds andfor boronated oligopeptides include among others: an alkylene chainhaving 1-20 and preferably 3-10 carbon atoms,

an alkyloxylene chain having 1-20 carbon and oxygen atoms and preferably5-12 carbon or oxygen atoms (an alkylene chain in which one or morenon-adjacent CH₂ moieties are replaced with —O—);an acetoxymethyl ether moiety;an alkylthiolene chain having 1-20 carbon and sulfur atoms andpreferably 5-12 carbon or sulfur atoms (an alkylene chain in which oneor more non-adjacent CH₂ moieties are replaced with —S—); oran alkylaminolene chain (i.e., a divalent alkylene chain in which one ormore non-adjacent CH₂ moieties are replaced with —NRc-) having 1-20carbon and nitrogen atoms and preferably 5-12 carbon or nitrogen atomswhere Rc is hydrogen or a C1-C3 alkyl group,a 5- or 6-member divalent alicyclic, heterocyclic, aryl or heteroarylring moiety, one or two alkylene moieties linked to a 5- or 6-memberalicyclic, heterocyclic, aryl or heteroaryl ring moiety.

In specific embodiments, phenylboronate compounds of this inventioncontain a reactive functional group for attachment of the phenylboronateto a nucleic acid, an amino acid, peptide or protein. The reactivefunctional group can, for example, be a group that reacts with an amine,a carbonyl, a carboxylate, a carboxylic ester, a sulfhydryl or ahydroxyl group. Preferably such reactive groups react to attach thephenylboronate to the nucleic acid, amino acid, peptide or protein underconditions such that the phenylboronate substantially retains sugarbinding activity and which do not substantially detrimentally affect thebiological activity of interest of the amino acid, peptide or protein.In a specific embodiment, the phenyl boronate contains an amine reactivegroup for bonding to an amine group of a nucleic acid base. A variety ofreactive groups useful for coupling to a nucleic acid, nucleoside,peptide or protein are known in the art and one of ordinary skill in theart can select among such known reactive groups to practice the methodsof the present invention without undo experimentation.

An overview of bioconjugation methods that can be employed forboronation of peptides and proteins is found in Hermanson, G. T.Bioconjugation Techniques (2^(nd) Ed.) 2008 Academic Press/ElsevierLondon, UK. This reference also contains detailed descriptions ofhomobifunctional and heterobifunctional crossing linking reagents whichcan be employed to covalently attach a phenylboronate group to an aminoacid, peptide or protein.

Amine-reactive groups are exemplified by a carboxylate group, acarboxylate ester group, an acid chloride group, an aldehyde group, anacyl azide group, an epoxide, an isothiocyanate group, an isocyanategroup, an imidoester group or an anhydride group. Amines react withcarboxylates in the presence of coupling reagents, such ascarbodiimides. Amine-reactive groups include active carboxylic acidester groups, such as succinimidyl ester groups or sulfosuccinimidylester groups (e.g., N—OH succinimidyl or N—OH sulfosuccinimidyl groups);haloalkyl ester groups, such as trifluoroalkyl ester groups andhexafluoroalkyl ester groups; halophenyl ester groups, particularlyfluorophenyl and chlorophenyl ester groups, including penta- andtetrafluorophenyl ester groups, pentachlorophenyl ester groups;nitrophenyl ester groups, including 2-nitrophenyl, 4-nitrophenyl and2,4-dinitrophenyl ester groups; as well as other substituted phenylester groups, including sulfodichlorophenol ester groups.

Examples of amine-reactive groups and their reactions with amino groupsare provided in FIG. 8. For example, carboxylate-substitutedphenylboronic acid can be coupled to an amino group of an amino acid,peptide or protein to form an amide linkage employing a coupling agent,such as a carbodiimide. A formyl-substituted phenylboronic acid can bereacted with an amino group of an amino acid, peptide or protein byreductive amination forming a —NH—CH₂— linkage. An acylazide-substitutedphenylboronic acid can be coupled to an amino group of an amino acid,peptide or protein to form an amide linkage. A phenylboronatesubstituted with an activated ester —COO—Z₁ reactive group can bereacted with an amino group of an amino acid, peptide or protein formingan amide linkage. A phenylboronate substituted with an isocyanate or aisothiocyanate reactive group can be reacted with an amino group of anamino acid, peptide or protein forming a urea or thiourea linkage,respectively. A phenylboronate substituted with an anhydride group, suchas a maleic anhydride group can be reacted with an amino group of anamino acid, peptide or protein forming an amide bond where at least aportion of the anhydride forms a spacer between the phenylboronate andthe amino group. An imidoester-substituted phenylboronate can be reactedwith an amino group of an amino acid, peptide or protein forming anamidine linkage. Reactions analogous to those shown in FIG. 8 can beused to couple the phenylboronate to an amine group of a nucleic acid.

General conditions for carrying out reactions between amine-reactivegroups and amino groups of a nucleic acid, an amino acid, peptide orprotein are well known in the art and can be carried out by one ofordinary skill in the art without undue experimentation. As illustratedin FIG. 8, the reactive group can be a substituent on the phenyl ring ofthe phenylboronate or can be contained in the substituent. The reactivegroups exemplified in FIG. 8 can also be linked to the phenyl ring ofthe phenylboronate by a spacer moiety L, such as described herein. Itwill be appreciated that it may be useful to protect the —B(OH)₂ groupof the phenylboronate while carrying out such reactions with aminogroups. Useful protecting groups for the —B(OH)₂ are known in the art,for example, pinacol, perfluoropinacol, pinanediol, ethylene glycol,diethylene glycol, catechol, 1,2-cyclohexanediol, 1-3-propanediol,2,3-butanediol, glycerol, neopentylglycol, diethanolamine,N-methyldiethanolamine, and 1-(4-methoxyphenyl)-2-methylpropane-1,2-diolcan be employed. See for example, Yan J. et al. (2005) [23].

Sulfhydryl-reactive groups are exemplified by haloacetyl andhaloacetamidyl groups, particularly iodoacetyl and bromoacetyl orcorresponding acetamidyl groups, maleimide groups, haloalkyl groups,halobenzyl groups, acryloyl groups, epoxide groups, groups that undergothiol-disulfide exchange, such as dipyridyl disulfide groups or2,2′-dihydroxy-6,6′-dinaphthyldisulfide groups, or thiosulfate groups.Exemplary sulfhydryl-reactive groups are provided in FIG. 9 where thelinkages formed are illustrated, for example —S—CH₂—CH₂—, —S—CH₂—CHR—,or —S—S— linkages can be formed (where R depends upon the reactive groupemployed). General conditions for carrying out reactions betweensulfhydryl-reactive groups and sulfhydryl groups of an amino acid,peptide or protein are well known in the art and can be carried out byone of ordinary skill in the art without undue experimentation. Asillustrated in FIG. 9, the reactive group can be a substituent on thephenyl ring of the phenylboronate or can be contained in thesubstituent. The reactive groups exemplified in FIG. 9 can also belinked to the phenyl ring of the phenylboronate by a spacer moiety L,such as described herein. It will be appreciated that it may be usefulto protect the —B(OH)₂ group of the phenylboronate while carrying outsuch reactions with sulfhydryl groups. Useful protecting groups for the—B(OH)₂ are known in the art as described hereinabove.

Carboxylate-reactive functional groups are exemplified by amines (e.g.,employing a carbodiimide), hydrazine groups, hydrazide groups,sulfonylhydrazide groups, diazoalkyl groups, diazoaryl groups,diazoacetyl groups, hydroxyl groups or sulfhydryl groups. FIG. 10illustrates coupling of an amino substituent on a phenylboronate with acarboxylic acid group of an amino acid, peptide or protein (P—COOH)employing a coupling reagent such as a carbodiimide. FIG. 10 alsoillustrates an alternative reaction where the carboxylate group isactivated, for example by formation of an active ester or by reactionwith carbonyl diimidazole to form an activated carbonyl followed byreaction with a hydroxyl group to form an ester, a sulfhydryl group toform a thioester or a hydrazine to form a hydrazide. As illustrated inFIG. 10, the carboxylate-reactive group can be linked to the phenyl ringof the phenylboronate by a spacer moiety L, such as described herein.Alternatively, the reactive group can be substituted directly on thephenyl ring of the phenylboronate. It will be appreciated that it may beuseful to protect the —B(OH)₂ group of the phenylboronate while carryingout such reactions with carboxylate groups. Useful protecting groups forthe —B(OH)₂ are known in the art as described hereinabove.

Hydroxyl-reactive functional groups are exemplified by isocyanategroups; epoxide groups; alkyl or aryl halide group, e.g., a halotritylgroup; an activated carbamate group, an activated ester group (such asdescribed above), N,N′-disuccinimidyl carbonate groups orN-hydroxysuccinimidyl chloroformate groups. FIG. 11 illustrates reactionof the hydroxyl group of a serine which may be in a peptide or proteinwith an isocyanate-substituted phenylboronate to form an —OCO—N—linkage. Terminal serines can be treated with periodate to generate analdehyde which can then be reacted with hydrazine groups, hydroxylaminegroups, or amine groups. The hydroxyl group (phenolate) of a tyrosineresidue can react with an acylating or alkylating agent forming an esteror ether, respectively, with a diazonium group to form a diazo compoundor with an amine group in the presence of an aldehyde (e.g.,formaldehyde or glutaraldehyde) to form a Mannich condensation productas also illustrated in FIG. 11. As shown in FIG. 11, the reactive groupcan be a substituent on the phenyl ring of the phenylboronate.Alternatively the hydroxyl-reactive group can also be linked to thephenyl ring of the phenylboronate by a spacer moiety L, such asdescribed herein. It will be appreciated that it may be useful toprotect the —B(OH)₂ group of the phenylboronate while carrying out suchreactions with carboxylate groups. Useful protecting groups for the—B(OH)₂ are known in the art as described hereinabove.

Aldehyde and ketone-reactive groups are exemplified by hydrazine groupsand derivatives thereof including hydrazides, semicarbazides andcarbohydrazides, and amino groups. Various methods for introduction ofaldehyde and ketone groups into amino acids, peptides and proteins areknown in the art. For example, N-terminal serine and threonines can beoxidized using periodate to form aldehyde groups. As illustrated in FIG.12, an aldehyde or ketone group of an amino acid, peptide or proteinreacts with a hydrazide group to form a hydrazine. Also as illustratedin FIG. 12, an aldehyde reacts with an amine to form a Mannichcondensation product.

Azide groups react with alkenyl or akynyl groups (in so-called Clickreactions) to form triazolines or triazoles.

Phosphinothioesters react with azide groups as described in U.S. Pat.Nos. 6,972,320, 7,256,259, and 7,317,129 and U.S. published applicationUS 2010/0048866 to form amide bonds in a traceless Staudinger ligation.Phosphinothioesters can be prepared employing phosphinothiol reagents asalso described in these references. Each of these references isincorporated by reference herein in its entirety for descriptions ofsuch ligation reactions, methods of making azides, and methods of makingphosphinothioesters.

Aldehyde, ketone, azide, activated esters groups, thioester, orphosphinothiol groups are introduced or generated in amino acids,peptides and proteins to be boronated by any art-known methods and in aspecific embodiment by reaction of a protein modifying reagent with oneor more of an amine group, a carboxylic acid group, a sulfhydryl groupor a hydroxyl group of an amino acid, peptide or protein.

Boronation methods of this invention can employ any art-known peptideligation method for attaching one or more boronated amino acids or oneor more boronated oligopeptides to a peptide or protein for enhancementof cellular uptake of the peptide or protein. Useful peptide ligationmethods include, among others, native chemical ligation, expressedprotein ligation, methods described by Offer and Dawson Org. Lett., 200,2, 23-26; Staudinger ligation methods, including those described in U.S.Pat. No. 6,972,320, U.S. Pat. No. 7,256,259 and U.S. Pat. No. 7,317,129,Garcia, et al. Tetrahedron Lett. 1984, 25, 4841-4844, Malkinson, L. P.;Falconer, R. A; Toth, I. J. Org. Chem. 2000, 65, 5249-5252 (and otherreferences cited in U.S. Pat. No. 6,972,320), and Saxon, E.; Bertozzi,C. R. Science 2000, 287, 2007-2010. Each of these references isincorporated by reference herein in its entirety for descriptions ofpeptide ligation methods.

Boronation methods of this invention can employ various bioconjugationmethods as known in the art. One or ordinary skill in the art in view ofthe descriptions herein and bioconjugation methods and peptide ligationmethods known in the art can conjugate one or more phenylboronatecompounds of this invention or one or more boronated oligopeptides ofthis invention to an amino acid, peptide or protein.

Boronation methods of this invention can employ crossing linkingreagents for conjugating phenylboronate compounds to amino acids,peptides or proteins or for conjugating boronated oligopeptides topeptides or proteins. Crosslinking agents effect conjugation of twoselected molecules and may also provide a spacer moiety between theconjugated molecules. Coupling agents, such as carbodiimides, arezero-length crosslinking reagents in which a single bond is formedbetween the molecules with no additional atoms added between themolecules as a spacer or linker. For example, a carbodiimide can be usedto conjugate a molecule carrying an amine group (i.e., an amine) to amolecule carrying a carboxylate group (a carboxylic acid) by formationof an amide bond with formal loss of H₂O.

Various carbodiimides are known in the art and a number are commerciallyavailable for conjugation including among others EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), DCC(dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), CMC(N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulfonate), Woodward's reagent K(2-ethyl-5-phenylisoxazolium-3′-sulfonate), and CDI (N,N′-carbonyldiimidazole), each of which is useful for conjugating an amine with acarboxylate. CDI can also be used for conjugating a hydroxyl group to anamine forming a carbamate.

Homobifunctional crosslinking reagents contain two identical reactivegroups separated by a spacer or linker moiety. Heterobifunctionalcrosslinking reagents contain two reactive groups with differentselectively for reaction, e.g., an amine-reactive group and asulfhydryl-reactive group separated by a spacer or linker moiety.Various homobifunctional and heterobifunctional crossing linkagereagents are known in the art and a number are commercially availablefrom Pierce (Thermo Scientific), Rockford, Ill., Sigma-Aldrich, St.Louis, Mo. or Molecular Probes (Life Technologies), Eugene Oreg.

Useful homobifunctional crosslinking reagents include those carrying twoamine-reactive groups, those carrying two sulfhydryl reactive groups,those carrying two carboxylate reactive groups, or those carrying twoaldehyde or ketone reactive groups.

Useful heterobifunctional crosslinking reagents include those carryingone of an amine-reactive group, a sulfhydryl reactive group, acarboxylate reactive group, or an aldehyde or ketone reactive group andone of a different reactive group selected from an amine-reactive group,a sulfhydryl reactive group, a carboxylate reactive group, or analdehyde or ketone reactive group.

Homobifunctional and heterobifunctional crosslinking reagents can ingeneral contain any spacer or linking moiety compatible with thereactive groups therein wherein the spacer or linker itself is notreactive with the compounds to be conjugated. In specific embodiments,the spacer or linking moiety typically ranges from 3-20 atoms (typicallyC, O, S and/or N atoms) in length (including residues from the reactivegroup), and optionally contain one or more carbon-carbon double bonds,and/or a 5- or 6-member alicyclic, heterocyclic, aryl or heteroarylring. Carbon atoms in the spacer or linker are often substituted withone or more hydroxyl groups, oxo moieties (═O), or halogens (e.g., F).Nitrogen groups in the linker may be substituted with hydrogen or withC1-C3 alkyl groups. The spacer or linker may contain one or two —S—S—and/or —SO₂— moieties which are cleavable. The spacer or linker may beselectively cleavable, for example, by change of conditions (e.g., pHchange), addition of a cleavage reagent, or photoirradiation (e.g., UVirradiation). In specific embodiments, a cleavable linker includes oneor two disulfide bonds which are selectively cleavable, for example ontreatment with dithiothreitol, a cleavable linker contains a diol moietywhich is selectively cleavable by treatment for example with periodate,an ester moiety, which id selectively cleavable by treatment withhydroxylamine, a sulfone moiety (—SO₂—) which is selectively cleavableunder alkaline conditions.

Homobifunctional crosslinking reagents can be used, for example, toconjugate an amine group of a nucleic acid, an amino acid, peptide orprotein with an amine substituent on a phenylboronate compound.Amine-reactive groups employed in homobifunctional crosslinking reagentsinclude among others, activated ester groups, such as NHS esters(N-hydroxysuccinimide esters) or sulfo NHS esters(N-hydroxysulfosuccinimide esters), imidoester group, such asmethylimidate salts, isothiocyanate groups and aryl halide groups, suchas difluorobenzene derivatives. Amine-reactive homobifunctional includeamong others: dithiobis(succinimidylproprionate) [DSP] and its sulfo-NHSanalog [DTSSP], disuccinimidyl suberate [DSS] and its sulfo-NHS analog[BS3], disuccinimidyl tartarate [DST] and its sulfo NHS analog[sulfo-DST], bis(2-succinimidyloxy-carbonyloxy)ethylsulfone [BSOCOES]and its sulfo-NHS analog [sulfo-BSOCOES], ethylene glycolbis(succinimidylsuccinate) [EGS] and its sulfo-NHS analog [sulfo-EGS],disuccinimidyl glutarate [DSG], N,N′disuccinimidyl carbonate [DSC],dimethyl adipimidate [DMA], dimethyl 3,3-dithiobispropionimidate [DTBP],4,4′-disiothiocyanatostilbene-2,2′-disulfonic acid salts,1,5-difluoro-2,4-dinitrobenzene [DFDNB],4,4′-difluoro-3,3′-dinitrodiphenylsulfone.

Homobifunctional crosslinking reagents can be used, for example, toconjugate a sulfhydryl group of an amino acid, peptide or protein with asulfhydryl substituent on a phenylboronate compound. Sulfhydryl-reactivegroups employed in homobifunctional crosslinking reagents include amongothers, alkyl halide groups, maleimide groups, and dithiopyridyl groups.Sulfhydryl-reactive homobifunctional crosslinking reagents include,among others, 1,4-di-[3′-(2′-pyridyldithio)propionamido]-butane [DPDPB],bismaleimidohexane[BMH], 1,4-bismaleimidyl-2,3-dihyroxybutane [BMDB],1,8-bismaleimidodiethyleneglycol [BM9PEG)₂], bismaleimidoethane [BMOE],dithiobismaleimidoethane [DTME], andN,N′-hexamethylene-bis(iodoacetamide).

Hydroxyl-reactive homobifunctional crosslinking reagents can be used toconjugate a hydroxyl group on an aminoacid, peptide or proteins with ahydroxyl group substituent on a phenylboronate compound.Hydroxyl-reactive groups include those having epoxide groups, such asdiglycidylethers, particularly 1,4-butanediol diglycidyl ether.

Bis-epoxide reagents can also react with amine groups and sulfhydrylgroups. A carboxylate group in an amino acid, peptide or protein can beconjugated to a carboxylate group substituent in a phenylboronatecompound, for example, by generating an active ester at the carboxylategroups and esterifying the active esters with an alkanediol crosslinkingreagent, such a 1,6-hexane diol, or 1,12-dodecanediol.

Aldehyde/ketone-reactive homobifunctional crosslinking reagents can beused to conjugate an aldehyde or ketone group introduced or generated inan amino acid, peptide or protein with an aldehyde or ketone groupsubstituent on a phenylboronate compound. Bis-hydrazide reagents can beused to crosslink molecules containing aldehyde or ketone groups,examples of such crosslinking reagents include among others adipic aciddihydrazide and carbohydrazide.

Heterobifunctional crosslinking reagents include those which contain anamine reactive group and a sulfhydryl-reactive group. For example, sucha heterobifunctional crosslinking reagent can be used to link an aminegroup in a nucleic acid, an amino acid, peptide or protein with asulfhydryl substituent in a phenylboronate or alternatively to link asulfhydryl group in an amino acid, peptide or protein with an aminesubstituent in a phenylboronate compound.

Exemplary heterobifunctional crosslinking reagents include thosecarrying an activated ester group, such as an NHS ester (or sulfo-NHSester) group or a nitrophenyl or other substituted phenyl ester and amaleimide group; those carrying such an activated ester group and adithiopyridyl group, those carrying an activated ester group and anhaloacetyl group (e.g., an iodoacetyl group), or those carrying animidoester group and a maleimide group.

Exemplary heterobifunctional amine/sulfhydryl-reactive crosslinkingreagents include, among others, N-(γ-maleimidobutyryloxy)succinimideester [GMBS] and its sulfo-NHS analog [sulfo-GMBS],4-succinimidyloxycarbonyl-α-(2-pyridyldithio)toluene [SMPT],succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [SMCC] andits sulfo-NHS analog [sulfo-SMCC],m-maleimidobenzoyl-N-hydroxy-succinimide ester [MBS] and its sulfo-HNSanalog [sulfo-MBS], N-succinimidyl(4-iodoacetyl)-aminobenzoate [SIAB]and its sulfo-HNS analog [sulfo-SIAB],succinimidyl-6-(iodoacetyl)aminohexanoate [SIAX],N-succinimidyl-3-(2-pyridylthio)propionate [SPDP],succinimidyl-4-(p-maleimidophenyl)butyrate [SMPB] and its sulfo-NHSanalog [sulfo-SMPB],succinimidyl-([N-maleimidopropionamidol]ethyleneglycol esters [SM(PEG)n,where n is 4, 6, 8, 12, 24] and p-nitrophenyl iodoacetate [NPIA],N-hydroxysuccinimidyl 2,3-dibromopropionate [SDBP].

Heterobifunctional crosslinking reagents useful in this invention forconjugation of an amino acid, peptide or protein with a phenylboronatecompound include those which contain a sulfhydryl-reactive group, suchas a maleimide or a pyridyldithio group, and a hydrazide which reactswith a carbonyl (aldehyde or ketone). Such reagents are particularlyuseful for linking a phenylboronate having an aldehyde of ketonesubstituent with an amino acid, peptide or protein having a sulfhydrylgroup. In specific embodiments, 4-(4-N-maleimidophenyl)butyric acidhydrazide HCl [MPBH],4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide [M₂C2H], and3-(2-pyridyldithio)propionyl hydrazide [PDPH] are such aheterobifunctional crosslinking reagents.

Heterobifunctional crosslinking reagents useful in this invention forconjugation of an amino acid, peptide or protein with a phenylboronatecompound include those which contain a sulfhydryl-reactive group, suchas a maleimide, and an isocyanate group for reaction with a hydroxylgroup. In a specific embodiment, N-(p-maleimidophenyl)-isocyanate issuch a heterobifunctional crosslinking reagent.

Heterobifunctional crosslinking reagents also include those whichcontain one of an amine-reactive, sulfhydryl-reactive,carboxylate-reactive or carbonyl-reactive group and a photoreactivegroup which is activated on irradiation to reactive with variousreactive groups, including nucleophiles, reactive hydrogen, activehydrogen amines or olefins. Such reagents are particularly useful forlinking an amino acid, peptide or protein having an amine group,sulfhydryl group, carboxylate group, or carbonyl group with aphenylboronate compound which reacts with the activated photoreactivegroup.

Photoreactive groups include, among others, optionally substituted arylazides which are photolyzed to aryl nitrenes or to dihydroazepinegroups, diazo or diazopyruvate groups which are photolyzed to reactivecarbines, diazirines which are photolyzed to reactive carbenes, andbenzophenone groups which are photolyzed to give a highly reactivetriplet-state ketone intermediate.

Aryl azides include, among others, phenyl azide groups, fluorinatedphenyl azide groups (e.g., tetrafluorophenyl azide groups),hydroxyphenyl azide groups (e.g., ortho or meta hydroxyphenylazidegroups), nitrophenylazide groups, or a 7-azido-4-methylcoumarin group.Aryl nitrenes can undergo addition reactions with double bonds, andinsertion reactions into active hydrogen bonds (C—H or N—H bonds).

Dehydroazepine intermediates react with nucleophiles by nucleophilicaddition, particularly with amines. The aryl group of the aryl azide orthe 7-member dehydroazepine ring becomes part of the linker formed oncrosslinking.

The triplet state intermediate formed on photolysis of benzophenonereacts with reactive hydrogen containing groups H—R′ to formR—C(OH)(Ph)₂.

Carbenes formed on photolysis of diazo compounds can insert into activeC—H or N—H bonds or add to double bonds. Carbenes formed on photolysisof diazopyruvate groups rearrange to reactive ketenes which reactivewith nucleophiles, including amines.

Specific examples of such photoreactive heterobifunctional crosslinkingreagents include, among others, N-hydroysuccinimidyl-4-azidosalicyclicacid [NHS-ASA] or its sulfo-NHS analog [sulfo-NHS-ASA],sulfosuccinimidyl-(4-azidosalicylamino)hexanoate [sulfo-NHS-LC-ASA],sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate[SAED], p-nitrophenyl diazopyruvate [pNPDP],p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate [PNP-DTP],1(p-azidosalicylamido)-4-(iodoacetamido)butane [ASIB],benzophenone-4-iodoacetamide, benzophenone-4-malemide, p-azidobenzoylhydrazide [ABH], and 4-(p-azidosalicylamido)butylamine [ASBA],N-(2-((2-((4-azido-2,3,5,6-tetrafluoro)benzoyl)amino)ethyl)dithio)ethyl)-maleimide[TFPAM-SS1],

With respect to any specific homo- or heterobifunctional crosslinkingreagents noted above, it will be appreciated that analog crosslinkingreagents having different lengths of linker moiety are also includedherein. Homobifunctional crosslinking reagents preferred for use in thepresent invention are amine-reactive or sulfhydryl-reactive crosslinkingreagents. Heterobifunctional crosslinking reagents preferred for use inthe present invention are those that contain an amine-reactive group anda sulfhydryl-reactive group.

It will be appreciated by one of ordinary skill in the art that thespecific coupling or crosslinking methods described herein can beadapted for use with any cargo molecule.

The invention provides a method for improved delivery of a cargomolecule nucleic acid, peptide or protein to a cell which comprises thestep of contacting the cell or tissue containing the cell with a peptideor protein boronated with a phenylboronic acid. Any methods known in theart for contacting a cell or tissue containing the cell can be employedwhich will bring the boronated cargo molecule into the vicinity of thecell or tissue. Contacting may occur in vitro by addition of a solutioncontaining the boronated cargo molecule to a solution or mediumcontaining or supporting the cell or tissue. Contacting may occur invivo by any method known in the art for administration of a solution orother composition containing the boronated peptide or protein to anorganism containing the cell or tissue.

Any suitable form of administration can be employed in the methodsherein. The compounds of this invention can, for example, beadministered orally, topically, intravenously, intraperitoneally,subcutaneously, or intramuscularly, in any suitable dosage forms wellknown to those of ordinary skill in the pharmaceutical arts. Theboronated cargo molecules are optionally administered with apharmaceutical carrier selected upon the basis of the chosen route ofadministration and standard pharmaceutical practice, such as, forexample, as described in Remingtons Pharmaceutical Sciences, 17thedition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa.(1985), which is incorporated herein by reference in its entirety forsuitable administration and carriers.

Cargo molecules include nucleic acids, peptides, proteins, smallmolecule drugs, reporters and labeling (fluorescent labels or isotopiclabels for example), imaging agents, contrast agents, particles carryingreactive functional groups, quantum dots carrying reactive functionalgroups, among others. In general any cargo molecule that is desired tointroduce into a cell can be employed in the methods of this invention.Cargo molecules include those having a biological activity. In specificembodiments, biological activity of interest of the cargo molecule isretained on boronation or is recovered on selective removal ofboronation after delivery to a cell. In a specific embodiment, theboronated cargo molecule retains at least 10% of a selected biologicalactivity of the cargo molecule prior to boronation. In other specificembodiments, the boronated cargo molecule retains at least 50% of aselected biological activity of the cargo molecule prior to boronation.In a further specific embodiment, the boronated cargo molecule retainsat least 80% of the activity of the cargo molecule prior to boronation.

In a specific embodiment, the cargo protein is an enzyme. In a specificembodiment, the cargo protein is not glycosylated (i.e., is not aglycoprotein). In a specific embodiment, the boronated cargo peptide orprotein retains at least 10% of a selected biological activity of theprotein prior to boronation. In other specific embodiments, theboronated cargo peptide or protein retains at least 50% of a selectedbiological activity of the protein prior to boronation. In a furtherspecific embodiment, the boronated cargo peptide or protein retains atleast 80% of the activity of the peptide or protein prior to boronation.Peptides and proteins include those having enzyme activity.

Cargo peptides include peptide ligands, cytotoxic peptides, bioactivepeptides, diagnostic agents, among others. Cargo peptides include thosehaving 2-1000 amino acids, 2-500 amino acids, 2-250 amino acids, 2-100amino acids, 2-50 amino acids, and 2-25 amino acids and 2-10 aminoacids.

Peptides and proteins include antibodies and functional fragmentsthereof, where the term antibody is used broadly herein. Morespecifically, antibodies include among others, monoclonal antibodiesincluding humanized antibodies, human antibodies, interspeciesantibodies, chimeric antibodies, human monoclonals, humanizedmonoclonals, interspecies antibodies made by any art-known methods.Functional fragments of antibodies include F(ab′)2, F(ab)₂, Fab′, Fab,Fv, among others, as well as hybrid fragments. Additionally, antibodiesinclude subfragments retaining the hypervariable, antigen-binding regionof an immunoglobulin and preferably having a size similar to or smallerthan a Fab′ fragment. Such fragments and subfragments including singlechain fragments or multiple chain fragments, which incorporate anantigen-binding site and exhibit antibody function, are known in the artand can be prepared by methods that are well-known in the art, includingby methods of preparing recombinant proteins. Antibodies and fragmentsthereof include therapeutic antibodies which are known in the art[Chames et al. 2009, 28]. This reference is incorporated by referenceherein in its entirety for descriptions of therapeutic antibodies whichcan be employed in the present invention.

In a specific embodiment, the cargo molecule is a nucleic acid which maybe RNA or DNA, or an analog of a nucleic acid which may be a peptidenucleic acid, a locked nucleic acid, or a phosphoradiamidate morpholinooligomer. Other art-known nucleic acid analogs include carbamate-linkedDNA, phosphorothioate-linked DNA, 2′-O-methyl RNA,phosphotriester-linked DNA or methylphosphonate-linked DNA. The cargonucleic acid can be single- or double-stranded. The nucleic acid can bean oligonucleotide or analog thereof having 2-100, 2-50 or 2-25 bases.The nucleic acid can be SiRna, microRNa, antisense oligonucleotides,decoy DNA, plasmids or other nucleic acid structures such asminicircles. Nucleic acids and analogs thereof are available fromcommercial sources, can be isolated from natural source or can beprepared by methods that are well-known in the art.

In a specific embodiment, the boronated cargo nucleic acid retains atleast 10% of a selected biological activity of the nucleic acid prior toboronation. In other specific embodiments, the boronated cargo nucleicacid retains at least 50% of a selected biological activity of thenucleic acid prior to boronation. In a further specific embodiment, theboronated cargo nucleic acid retains at least 80% of the activity of thenucleic acid prior to boronation. In a specific embodiment, thebiological activity of the nucleic acid that is retained in binding to acomplementary nucleic acid or binding to another biological molecule(e.g., a peptide or protein).

Cargo nucleic acids include those having 2-1000 bases, 2-500 bases,2-250 bases, 2-100 bases, 2-50 bases, and 2-25 bases and 2-10 bases.Nucleic acids include nucleosides and analogs thereof.

An aliphatic group as used herein refers to a monovalent non-aromatichydrocarbon group which include straight chain, branched, or cyclichydrocarbon groups which can be saturated or unsaturated with one ormore double bonds or one or more triple bonds. Aliphatic groups maycontain portions which are straight-chain or branched in combinationwith one or more carbon rings. Carbon rings of aliphatic groups maycontain one or more double bonds or one or more triple bonds. Carbonrings of aliphatic groups can contain 3- to 10-membered rings. Suchcarbon rings may be fused and may be bicyclic or tricyclic. Aliphaticgroups are optionally substituted with one or more non-hydrogensubstituents where optional substituents are described herein. Unlessotherwise specified, an aliphatic group can contain 1-20 carbon atoms orcan contain 1-10 carbon atoms. Aliphatic groups include those containing1-3, 1-6, and 1-8 carbon atoms. Aliphatic groups include, among others,alicyclic groups, alkyl groups, alkenyl groups and alkynyl groups

An alicylic group as used herein refers to a monovalent non-aromaticcyclic hydrocarbon group which can be saturated or unsaturated with oneor more double bonds or one or more triple bonds. Alicyclic ringsinclude those containing 3- to 10-membered carbon rings. Alicyclicgroups include those containing one, two, three or more rings which maybe fused or linked by straight chain or branched alkylene, alkenylene oralkynylene moieties. Alicyclic groups include bicyclic and tricyclicrings. Alicyclic groups include those in which one or more carbon ringsare substituted with a straight-chain or branched alkyl, alkenyl oralkynyl group. To satisfy valence requirements, a ring atom may besubstituted with hydrogen or optionally with non-hydrogen substituentsas described herein. One or more carbons in an alicyclic group can be—CO— groups, i.e. a carbon can be substituted with an oxo (═O) moiety.Alicyclic groups are optionally substituted with one or morenon-hydrogen substituents where optional substituents are describedherein. Unless otherwise specified, an alicyclic group can contain 3-20carbon atoms or can contain 3-12 carbon atoms. Alicyclic groups includethose containing 3-6 and 3-8 carbon atoms. Alicyclic groups includeamong others cycloalkyl, cycloalkenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl andcyclohexadienyl groups, all of which are optionally substituted.

A heterocyclic group as used herein refers to a monovalent non-aromaticcyclic hydrocarbon group wherein one or more of the rings contain one ormore heteroatoms (e.g., N, S, O, or P) which rings can be saturated orunsaturated with one or more double bonds or one or more triple bonds.In specific embodiments of this invention, heterocyclic rings which aresubstituents of the compounds of formulas IA and IB do not contain boronatoms. Heterocyclic rings include those containing 3- to 10-memberedrings where 1, 2 or 3 of the ring members are heteroatoms. Heterocyclicgroups include those containing one, two, three or more rings which maybe fused or linked by straight chain or branched alkylene, alkenylene oralkynylene moieties. Heterocyclic groups include bicyclic and tricyclicgroups. Heterocyclic groups include those in which a heterocyclic ringis substituted with a straight-chain or branched alkyl, alkenyl oralkynyl group. To satisfy valence requirements, a ring atom may besubstituted with hydrogen or optionally with non-hydrogen substituentsas described herein. One or more carbons in a heterocyclic group can be—CO— groups. One or more carbons in a heterocyclic ring can be—CO-groups. Heterocyclic groups are optionally substituted with one ormore non-hydrogen substituents where optional substituents are describedherein. Ring carbons and, where chemically feasible, ring heteroatomsare optionally substituted. Unless otherwise specified, a heterocyclicgroup can contain 3-20 carbon atoms, can contain 3-12 carbon atoms orcan contain 3-6 carbon atoms. Heterocyclic groups include thosecontaining one or two 4-, 5- or 6-member rings at least one of which hasone, two or three N, O or S atoms and wherein a ring optionally has oneor two double bonds. Heterocyclic groups include those containing asingle 5- or 6-member ring having one, two or three N, O or S atoms andoptionally having one or two double bonds. Heterocyclic groups includethose having 5- and 6-member rings with one or two nitrogens and one ortwo double bonds. Heterocyclic groups include those having 5- and6-member rings with an oxygen or a sulfur and one or two double bonds.Heterocyclic groups include those having 5- or 6-member rings and twodifferent heteroatom, e.g., N and O, O and S or N and S. Heterocyclicgroups include those having 5- or 6-member rings and a singleheteroatom, e.g., N S or O. In specific embodiments, heterocyclic groupsdo not include any boron atoms. Specific heterocyclic groups includeamong others among others, pyrrolidinyl, piperidyl, piperazinyl,pyrrolyl, pyrrolinyl, furyl, thienyl, morpholinyl, oxazolyl, oxazolinyl,oxazolidinyl, indolyl, triazoly, and triazinyl groups, all of which areoptionally substituted.

In embodiments herein alicyclic or heterocyclic rings can be formedbetween certain substitution sites on the molecules of formulas IA orIB. Such rings include the atom(s) of or between the sites ofsubstitution and are defined with respect to the optional presence ofheteroatoms, the optional presence of —CO— moieties and the optionalpresence of double bonds as are alicylic and heterocyclic groups. Unlessotherwise specified such rings can contain 5-10-member rings and morepreferably contain 5- to 8-member rings and more preferably 5- or6-member rings. Ring atoms are optionally substituted as describedherein.

Aryl groups are monovalent groups containing at least one aromatic ring.Aryl groups include groups having one or more 5- or 6-member aromaticrings. Aryl groups can contain one, two or three, 6-member aromaticrings. Aryl groups can contain two or more fused aromatic rings. Arylgroups can contain two or three fused aromatic rings. Aryl groups maycontain one or more non-aromatic alicyclic rings in addition to anaromatic ring. Aryl groups are optionally substituted with one or morenon-hydrogen substituents as described herein. Substituted aryl groupsinclude among others those which are substituted with alkyl or alkenylgroups, which groups in turn can be optionally substituted. Specificaryl groups include phenyl groups, biphenyl groups, and naphthyl groups,all of which are optionally substituted as described herein. In aspecific embodiment, aryl groups are not substituted with aboron-containing substituent, e.g., —B(OH)₂ or a —CH₂—B—O— moiety.Substituted aryl groups include fully halogenated or semihalogenatedaryl groups, such as aryl groups having one or more hydrogens replacedwith one or more fluorine atoms, chlorine atoms, bromine atoms and/oriodine atoms. Substituted aryl groups include fully fluorinated orsemifluorinated aryl groups, such as aryl groups having one or morehydrogens replaced with one or more fluorine atoms. Unless otherwisespecified, an aryl group can contain 5-20 carbon atoms or can contain6-14 carbon atoms. Aryl groups also include those containing 6-12 carbonatoms.

Heteroaryl groups are monovalent groups having one or more aromaticrings in which at least one ring contains a heteroatom (a non-carbonring atom). Heteroaryl groups include those having one or twoheteroaromatic rings carrying 1, 2 or 3 heteroatoms and optionallyhaving one 6-member aromatic ring. Heteroaryl groups can contain 5-20,5-12 or 5-10 ring atoms. Heteroaryl groups include those having at leastone aromatic ring containing a heteroatom and one or two alicyclic,heterocyclic or aryl ring groups. Heteroaryl groups include those havingone aromatic ring containing a heteroatom and one aromatic ringcontaining carbon ring atoms. Heteroaryl groups include those having oneor more 5- or 6-member aromatic heteroaromatic rings and one or more6-member carbon aromatic rings. Heteroaromatic rings can include one ormore N, O, or S atoms in the ring. Heteroaromatic rings can includethose with one, two or three N, those with one or two 0, and those withone or two S, or combinations of one or two or three N, O or S. Specificheteroaryl groups include pyridinyl, pyrazinyl, pyrimidinyl, quinolinyl,and purinyl groups. In specific embodiment, heteroaromatic rings thatare substituents on compounds of formulas IA or IB do not contain Batoms.

In embodiments herein aryl or heteroaryl rings can be formed betweencertain substitution sites on the molecules of formulas IA or IB. Suchrings include the atom(s) of or between the sites of substitution andare defined with respect to the optional presence of heteroatoms. Unlessotherwise specified such rings can contain 5- or 6-member rings and ringatoms are optionally substituted as defined herein.

Alkyl groups are monovalent groups and include straight-chain, branchedand cyclic alkyl groups. Unless otherwise indicated alkyl groups includethose having from 1 to 20 carbon atoms. Alkyl groups include alkylgroups having 1 to 3 carbon atoms, alkyl groups having from 4-7 carbonatoms and alkyl groups having 8 or more carbon atoms. Cyclic alkylgroups include those having one or more rings. Cyclic alkyl groupsinclude those which have 1, 2 or 3 rings. Cyclic alkyl groups alsoinclude those having 3-10 carbon atoms. Cyclic alkyl groups includethose having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6-, 7-, or 8-member ring. Thecarbon rings in cyclic alkyl groups can also carry straight-chain orbranched alkyl group substituents. Cyclic alkyl groups can includebicyclic and tricyclic alkyl groups. Alkyl groups are optionallysubstituted with one or more non-hydrogen substituents as describedherein. Specific alkyl groups include methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl,n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl,cyclohexyl, decalinyl, and norbornyl, all of which are optionallysubstituted. Substituted alkyl groups include fully halogenated orsemihalogenated alkyl groups, such as alkyl groups having one or morehydrogens replaced with one or more fluorine atoms, chlorine atoms,bromine atoms and/or iodine atoms. Substituted alkyl groups includefully fluorinated or semifluorinated alkyl groups. Substituted alkylgroup include alkyl group substituted with one or more hydroxyl groups.Substituted alkyl groups include groups substituted with two or morehydroxyl groups, particularly where two hydroxyl groups are substitutedon adjacent carbon atoms.

Arylalkyl groups are monovalent alkyl groups substituted with one ormore aryl groups wherein the alkyl groups optionally carry additionalsubstituents and the aryl groups are optionally substituted. Specificarylakyl groups are phenyl-substituted alkyl groups, e.g., benzyl groupsor phenethyl groups which are optionally substituted. Heteroarylalkylgroups are monovalent alkyl groups substituted with one or moreheteroaryl groups wherein the alkyl groups optionally carry additionalsubstituents and the aryl groups are optionally substituted. Alkylarylgroups are monovalent aryl groups substituted with one or more alkylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are further optionally substituted. Specificalkylaryl groups are alkyl-substituted phenyl groups such as o-, m- orp-tolyl groups which are optionally substituted. Alkylheteroaryl groupsare monovalent alkyl groups substituted with one or more heteroarylgroups wherein the alkyl groups optionally carry additional substituentsand the heteroaryl groups are optionally substituted.

Alkenyl groups include monovalent straight-chain, branched and cyclicalkenyl groups which contain one or more carbon-carbon double bonds.Unless otherwise indicated alkenyl groups include those having from 2 to20 carbon atoms. Alkenyl groups include those having 2 to 4 carbon atomsand those having from 5-8 carbon atoms. Cyclic alkenyl groups includethose having one or more rings wherein at least one ring contains adouble bond. Cyclic alkenyl groups include those which have 1, 2 or 3rings wherein at least one ring contains a double bond. Cyclic alkenylgroups also include those having 3-10 carbon atoms. Cyclic alkenylgroups include those having a 5-, 6-, 7-, 8-, 9- or 10-member carbonring and particularly those having a 5- or 6-member ring. The carbonrings in cyclic alkenyl groups can also carry straight-chain or branchedalkyl or alkenyl group substituents. Cyclic alkenyl groups can includebicyclic and tricyclic alkyl groups wherein at least one ring contains adouble bond. Alkenyl groups are optionally substituted with one or morenon-hydrogen substituents as described herein. Specific alkenyl groupsinclude ethylene, propenyl, cyclopropenyl, butenyl, cyclobutenyl,pentenyl, pentadienyl, cyclopentenyl, cyclopentadienyl, hexylenyl,hexadienyl, cyclohexenyl, cyclohexadienyl, including all isomers thereofand all of which are optionally substituted. Substituted alkenyl groupsinclude fully halogenated or semihalogenated alkenyl groups.

Alkynyl groups include mono-valent straight-chain, branched and cyclicalkynyl group which contain one or more carbon-carbon triple bonds.Unless otherwise indicated alkynyl groups include those having from 2 to20 carbon atoms. Alkynyl groups include those having 2 to 4 carbon atomsand those having from 5-8 carbon atoms. Cyclic alkynyl groups includethose having one or more rings wherein at least one ring contains atriple bond. Cyclic alkynyl groups include those which have 1, 2 or 3rings wherein at least one ring contains a triple bond. Cyclic alkynylgroups also include those having 3-10 carbon atoms. Cyclic alkynylgroups include those having a 5-, 6-, 7-, 8-, 9- or 10-member carbonring and particularly those having a 5- or 6-member ring. The carbonrings in cyclic alkynyl groups can also carry straight-chain or branchedalkyl, alkenyl or alkynyl group substituents. Cyclic alkynyl groups caninclude bicyclic and tricyclic alkyl groups wherein at least one ringcontains a triple bond. Alkynyl groups are optionally substituted withone or more non-hydrogen substituents as described herein.

An alkoxy group is an alkyl group (including cycloalkyl), as broadlydiscussed above, linked to oxygen, a monovalent —O-alkyl group. Anaryloxy group is an aryl group, as discussed above, linked to an oxygen,a monovalent —O-aryl. A heteroaryloxy group is a heteroaryl group asdiscussed above linked to an oxygen, a monovalent —O-heteroaryl.Alkenoxy, alkynoxy, alicycloxy, heterocycloxy groups are analogouslydefined. All of such groups are optionally substituted.

The number of carbon atoms in a given group, such as an alkyl group, canbe indicated herein using the expression “Cm” where m is the number ofcarbon atoms. Thus, the expression “Cm1-Cm2” modifying a given chemicalgroup indicates that the group can contain from m1 to m2 carbon atoms.For example, a C1-C6 alkyl group contains 1 to 6 carbon atoms, exclusiveof carbons in any substituent on the alkyl group. Similar expressionscan be used to indicate the number of atoms of N (nitrogen), O (oxygen)or other elements in a given group.

As to any of the above groups which contain one or more substituents, itis understood, that such groups do not contain any substitution orsubstitution patterns which are sterically impractical and/orsynthetically non-feasible. In addition, the compounds of this inventioninclude all stereochemical isomers arising from the substitution ofthese compounds.

Phenylboronate compounds and boronated oligopeptides of the inventionmay be in the form of salts. Preferred salts are those that arebiologically acceptable for ultimate applications of boronated peptidesand proteins and which do not substantially detrimentally affect theeffectiveness and properties of the free bases or free acids, and whichare not biologically or otherwise undesirable. Salts may be preparedfrom addition of an organic or inorganic base to the free acid oraddition of an organic or inorganic acid to the free base.

Exemplary salts of free bases are formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, preferably hydrochloric acid, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, lactic acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, trifluoroacetic acid,N-acetylcysteine and the like.

Exemplary salts of free acids are formed with inorganic base include,but are not limited to, alkali metal salts (e.g., Li+, Na+, K+),alkaline earth metal salts (e.g., Ca2+, Mg2+), non-toxic heavy metalsalts and ammonium (NH4+) and substituted ammonium (N(R′)4+ salts, whereR′ is hydrogen, alkyl, or substituted alkyl, i.e., including, methyl,ethyl, or hydroxyethyl, specifically, trimethyl ammonium, triethylammonium, and triethanol ammonium salts), salts of cationic forms oflysine, arginine, N-ethylpiperidine, piperidine, and the like. Compoundsof the invention can also be present in the form of zwitterions.

The term kit refers to kits for enhancement of cellular delivery and tokits for boronating cargo molecules, particularly cargo molecules whichare nucleic acids, peptides or proteins. In one embodiment, kits of thisinvention include one or more of the phenylboronate compounds of thepresent invention or mixtures thereof and optionally reagents forligating, conjugating or reacting the phenylboronate compound with acargo molecule, e.g., a nucleic acid, peptide or protein to effectboronation of the cargo molecule. In another embodiment, kits of thisinvention include one or more boronated oligopeptides of this inventionand optionally reagents, such as one or more homo- or heterobifunctionalcrosslinking reagents, for ligating or conjugating the boronatedoligopeptide to a cargo molecule to effect boronation of the cargomolecule. In yet another embodiment, kits of this invention may containone or more protected boronated amino acids and optionally one or morenon-boronated amino acids suitable for carrying out solid phase peptidesynthesis of a boronated peptide including a boronated oligopeptide. Inthis embodiment, kits may also contain resin for carrying out solidphase peptide synthesis as well as reagents, solvents and othercomponents needed for or useful for carrying out solid phase peptidesynthesis.

Additionally such kits for synthesis of boronated oligopeptidesoptionally contain one or more reagents, such one or more couplingreagents, or one or more homo- or heterobifunctional crosslinkingreagents, for conjugation of the phenylboronate or boronatedoligopeptide to a peptide or protein to effect boronation of the peptideor protein. Kits of the invention may also contain reagents for labelingof cargo molecules including reagents for labeling nucleic acids,peptides and proteins in addition to boronation of cargo molecules. Kitsfor enhancing cellular uptake of a cargo molecule may further containone or more cargo molecules which are to be boronated for delivery tocells. Cargo molecules may further contain structural portions includingcell penetrating peptides or targeting peptides, such as nuclearlocalization signals (such as are known in the art). Such kits mayadditionally comprise cells and cell growth media.

Kits of the invention may comprise a carrier being compartmentalized toreceive in close confinement one or more containers, such as vials, testtubes, ampules, bottles and the like. Each of such container meanscomprises components or a mixture of components needed to perform theindicated boronation, solid phase synthesis of boronated oligopeptide,or enhancement of cellular uptake. The kits of the invention may furthercomprise one or more additional components (e.g., reagents and/orcompounds) necessary or desirable for carrying out one or moreparticular applications of the compositions of the present invention. Ingeneral kits may also contain one or more buffers, control samples,carriers or recipients, vessels for carrying out one or more reactions,vessels for containing cells and the like, one or more additionalcompositions of the invention, one or more sets of instructions, and thelike.

Bovine pancreatic ribonuclease (RNase A) is a small, well-characterizedenzyme that has been the object of much seminal work in proteinchemistry[9] If this ribonuclease can gain access to the RNA thatresides in the cytosol, then its prodigious catalytic activity can leadto cell death[10] Hence, RNase A can serve as an ideal model forassessing the delivery of a protein into the cytosol (rather than anendosome) because success can be discerned with assays of cytotoxicactivity.

Initially, the affinity of simple phenylboronic acids to certainsaccharides (e.g., D-fructose, D-glucose and Neu5Ac) was assessed.Sialic acid is of particular interest because of its abundance in theglycocalyx of cancerous cells. [11] Phenylboronic acid (PBA) binds withhigher affinity to sialic acid than to other pyranose saccharides. [12]suggesting that simple boronic acids could target chemotherapeuticagents selectively to tumors. Benzoboroxole(2-hydroxymethylphenylboronic acid) has the highest reported affinityfor pyranose saccharides [12-13] which are abundant in the glycocalyx.¹H NMR spectroscopy was used to evaluate the affinity of PBA andbenzoboroxole for fructose, glucose, and N-acetylneuraminic acid(Neu5Ac), which contains a sialic acid moiety, under physiologicalconditions. The Ka values determined (Table 1) are in gratifyingagreement with values determined by other workers using alternativeassays. [12a, 12c, 13] Both benzoboroxole and PBA have greater affinityfor Neu5Ac than for glucose and benzoboroxole has greater affinity thanPBA for each saccharide in the panel studied.

Initial studies of boronation of RNase employed benzoboroxole. Todisplay benzoboroxole moieties on RNase A, 5-amino2-hydroxymethylphenylboronic acid (1) was conjugated to protein carboxylgroups of the protein by condensation using a carbodiimide (Scheme 1).Of the 11 carboxyl groups of RNase A, 7.5±2.0 were condensed withboronate 1, as determined by mass spectrometry.

As an initial test of the affinity of boronated protein (boronated RNAseA) for oligosaccharides. The retention of boronated and unmodified RNaseA on a column of heparin, a common physiological polysaccharide, wascompared. Benzoboroxole-boronated RNase A was indeed retained longer onthe column (FIG. 2). The prolonged retention is believed due toboron-saccharide complexation. Addition of fructose in the elutionbuffer (0.10M fructose) diminished the retention of boronated RNAse A.This result is consistent with boron-saccharide complex formationindicating that fructose competes with immobilized heparin for boroncomplexation. To evaluate the enhanced affinity of boronated RNase A foroligosaccharides, its affinity for ganglioside GD3 within a1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposome was measured.This ganglioside has two sialic acid residues and is overexpressed onthe surface of cancer cells. [15] By using fluorescence polarization toanalyze binding, it was demonstrated that boronation increased theaffinity of the protein for the ganglioside, an effect that wasabrogated by fructose (FIG. 3). The Kd value of boronated protein forGD3 ganglioside liposomes was (54±11) M. This affinity is 440 foldgreater than that for the binding of a single benzoboroxole to Neu5Ac(Table 1), consistent with a multivalent interaction between theboronated protein and the ganglioside.

To quantify cellular internalization, fluorophore-labeled protein andflow cytometry was used. To determine concurrently if the pendantboronates elicited selectivity for cells with higher quantities ofcell-surface sialic acid, two lines of Chinese hamster ovary cells werecompared. Lec-2 cells have lower levels of sialic acid in theirglycocalyx than their progenitor line, Pro 5. [16] Boronation of RNase Aincreased cellular uptake in both cell lines by 4- to 5-fold (FIG. 4A).This enhancement in cell uptake was eliminated by addition of fructose.Cell-surface sialic acid-content was not observed to affect uptakesignificantly. Confocal microscopy of boronated protein demonstratedpunctate staining (FIG. 4B), which is consistent with uptake byendocytosis following complexation with cell-surface saccharides.

Although flow cytometry can quantify protein internalization into acell, it does not differentiate between proteins in endosomes versusthose in the cytosol. Delivery into the cytosol is important for theefficacy of numerous putative chemotherapeutic agents.

Boronated RNase A retained (17±2) % of its ribonucleolytic activity.[17] Accordingly, boronated RNase A has the potential to be cytotoxic,if it can enter the cytosol. Boronated RNase A inhibited theproliferation of a line of human erythroleukemia cells (K-562; FIG. 5).The addition of fructose diminished cytotoxic activity, presumably bydecreasing overall internalization. In contrast, chemically inactivated,boronated RNase A was much less cytotoxic, indicating thatribonucleolytic activity induced toxicity, not the pendant boronates.These results show that boronation not only facilitates cellular uptakeof an enzyme, but also allows for its delivery to the cytosol and themaintenance of its catalytic activity.

Boronates have attributes that make them attractive as mediators of drugdelivery. First, endosomes become more acidic as they mature. Insynergy, the affinity of boronates for saccharides decreases withdecreasing pH [12a]. Moreover, the ensuing loss of complexation causesboronates to become more hydrophobic[18] These attributes are believedto facilitate translocation of proteins with pendant boronates to thecytosol. Second, boronates are not cationic [19], averting thenon-specific Coulombic interactions elicited by cationic domains [2],which can lead to high rates of glomerular filtration andopsonization[20] Finally, numerous diseases are associated with changesin cell-surface glycosylation [11, 21]. Thus, boronic acids withspecificity for particular glycans can serve as the basis for targeteddelivery strategies[22]

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art, insome cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claim.

When a group of substituents is disclosed herein, it is understood thatall individual members of those groups and all subgroups, including anyisomers and enantiomers of the group members, and classes of compoundsthat can be formed using the substituents are disclosed separately. Whena compound is claimed, it should be understood that compounds known inthe art including the compounds disclosed in the references disclosedherein are not intended to be included. When a Markush group or othergrouping is used herein, all individual members of the group and allcombinations and subcombinations possible of the group are intended tobe individually included in the disclosure.

Every formulation or combination of components described or exemplifiedcan be used to practice the invention, unless otherwise stated. Specificnames of compounds are intended to be exemplary, as it is known that oneof ordinary skill in the art can name the same compounds differently.When a compound is described herein such that a particular isomer orenantiomer of the compound is not specified, for example, in a formulaor in a chemical name, that description is intended to include eachisomer, e.g., cis and trans isomers, and each enantiomer or diastereomerof the compound described individual or in any combination.

Compounds of the invention, and salts thereof, may exist in theirtautomeric form, in which hydrogen atoms are transposed to other partsof the molecules and the chemical bonds between the atoms of themolecules are consequently rearranged. It should be understood that alltautomeric forms, that may exist, are included within the invention.

The processes for preparation of the compounds herein can use mixturesof isomers, racemates, enantiomers, or diastereomers as startingmaterials and may result in mixtures of isomers, enantiomers ordiastereomers. If desired such mixtures can be separated by conventionalmethods, for example, by various chromatographic methods or fractionalcrystallization. Compounds of the invention may be in the free orhydrate form.

One of ordinary skill in the art will appreciate that methods, deviceelements, starting materials, and synthetic methods other than thosespecifically exemplified can be employed in the practice of theinvention without resort to undue experimentation. All art-knownfunctional equivalents, of any such methods, device elements, startingmaterials, and synthetic methods are intended to be included in thisinvention. Whenever a range is given in the specification, for example,a temperature range, a time range, or a composition range, allintermediate ranges and subranges, as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In boronation methodsherein the term consisting essentially of excludes any component notrequired for successful boronation. In methods of enhancing cellularuptake, the term consisting essentially of excludes any component notrequired for successful enhancement of uptake of a given peptide orprotein. Any recitation herein of the term “comprising”, particularly ina description of components of a composition or in a description ofelements of a device, is understood to encompass those compositions andmethods consisting essentially of and consisting of the recitedcomponents or elements. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.

Without wishing to be bound by any particular theory, there can bediscussion herein of beliefs or understandings of underlying principlesrelating to the invention. It is recognized that regardless of theultimate correctness of any mechanistic explanation or hypothesis, anembodiment of the invention can nonetheless be operative and useful.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

THE EXAMPLES Example 1 Material and Methods Materials:

N-Acetylneuraminic acid was from Carbosynth (Berkshire, UK).Phenylboronic acid, 2-hydroxymethylphenylboronic acid, and5-amino-2-hydroxymethylphenylboronic acid were from Combi-Blocks (SanDiego, Calif.). BODIPY® FL, STP ester was from Molecular Probes (Eugene,Oreg.). [methyl-3H] Thymidine (6.7 Ci/mmol) was from Perkin-Elmer(Boston, Mass.). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) and wild-type RNase A (Type III-A) were fromSigma-Aldrich (St. Louis, Mo.) and used without further purification.Ribonuclease substrate 6-FAM-dArUdAdA-6-TAMRA was from Integrated DNATechnologies (Coralville, Iowa). Bovine Serum Albumin (BSA) was fromThermo Scientific (Rockfield, Ill.). Lysozyme, from chicken egg white(Sigma L6876) was from Sigma-Aldrich (St. Louis, Mo.) and used withoutfurther purification. Biotinylated peptide was purchased and customsynthesized from Biomatik (Cambridge, Ontario, Canada). NeutrAvidin,Fluorescein conjugated (Thermo 31006) was from Pierce Protein ResearchProducts, Thermo Scientific (Rockford, III.). GD3 Ganglioside (bovinemilk; ammonium salt), 1,2 dioleoyl-sn-glycero-3-phosphocholine (DOPC),and an extruder were from Avanti Polar Lipids (Alabaster, Ala.). 16S—and 23S-Ribosomal (rRNA) from E. coli MRE600 was from Roche AppliedScience (Mannheim, Germany).

MES buffer was from Sigma-Aldrich and purified by anion-exchangechromatography to remove trace amounts of oligomeric vinylsulfonic acid.(B. D. Smith, M. B. Soellner, R. T. Raines, J. Biol. Chem. 2003, 278,20934-20938.)

Spectra/Por® dialysis bags (3500 MWCO) were from Fisher Scientific(Thermo Fisher Scientific, Walham, Mass.). Escherichia coli BL21 (DE3)cells were from Novagen (Madison, Wis.). Heparin HP column proteinpurification and analytical columns were from GE Biosciences(Piscataway, N.J.). Non-binding surface (NBS) 96-well plates were fromCorning (Corning, N.Y.). Terrific Broth (TB) was from Research ProductsInternational Corp (Mt. Prospect, Ill.). SDS-PAGE gels were from Bio-RadLaboratories (Hercules, Calif.).

Cell culture medium and supplements were from Invitrogen (Carlsbad,Calif.). Phosphate-buffered saline was either Dulbecco's PBS (DPBS) fromInvitrogen or the same solution made in the laboratory (PBS), containing(in 1.0 L): 0.2 g KCl, 0.2 g KH₂PO₄, 8 g NaCl, and 2.16 g Na₂HPO₄*7H₂Oat pH 7.4. All other chemicals used were of commercial reagent grade orbetter, and were used without further purification.

Instrumentation and Statistics

¹H NMR spectra were acquired at the National Magnetic Resonance Facilityat Madison at 298 K on an Avance III 500 MHz spectrometer with a TCI 500H—C/N-D cryogenic probe from Bruker AXS (Madison, Wis., ¹H, 500 MHz).Protein absorbance values were measured on a Varian Cary 50 UV-VisSpectrometer (Agilent Technologies, Santa Clara, Calif.) and/or aNanoVue spectrometer (GE Healthcare, Piscataway, N.J.). Confocalmicroscopy was carried out using an Eclipse C1 laser scanning confocalmicroscope from Nikon (Melville, N.Y.). Flow cytometry was done using aLSRII (BD Biosciences, San Jose, Calif.) at the University ofWisconsin-Madison Carbone Cancer Center Flow Cytometry Facility. Themass of proteins (RNase A, lysozyme, etc.), protein-conjugates, peptidesand boronated peptides were confirmed at the University ofWisconsin-Madison Biophysics Instrumentation Facility by matrix-assistedlaser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometrywith a Voyager-DE-PRO Biospectrometry Workstation from AppliedBiosystems (Foster City, Calif.). [methyl-³H] Thymidine incorporationinto K-562 genomic DNA was quantified by scintillation counting using aMicrobeta TriLux liquid scintillation and luminescence counter fromPerkin-Elmer. Fluorescence measurements were made with an infinite M1000plate reader from Tecan (Mannedorf, Switzerland). Calculations forstatistical significance were performed with GraphPad Prism version 5.02software from GraphPad Software (La Jolla, Calif.), and a value ofp<0.05 was considered to be significant.

Example 2 Determination of K_(a) Values by ¹H NMR Spectroscopy

Methodology to determine the values of K_(a) for boronic acids andsaccharides was adapted from work by Hall and coworkers. [M. Dowlut, G.Dennis, J. Am. Chem. Soc. 2006, 128, 4226-4227; M. Bérubé, M. Dowlut, D.G. Hall, J. Org. Chem. 2008, 73, 6471-6479]

A boronic acid (B) and a saccharide (S) were assumed to bind in onemodality, B·S:

B + S ⇄ B ⋅ S$K_{a} = \frac{\left\lbrack {B \cdot S} \right\rbrack}{\lbrack B\rbrack \lbrack S\rbrack}$

The [B·S]/[B] ratio was determined by the integration of aryl protons ofthe boronic acid.saccharide complex and the free boronic acid. Theindividual [B], [B·S], and [S] can be calculated from eq. 1-3.

$\begin{matrix}{{{\left\lbrack {B \cdot S} \right\rbrack + \lbrack B\rbrack} = \left\lbrack B_{T} \right\rbrack}{{\frac{\left\lbrack {B \cdot S} \right\rbrack}{\lbrack B\rbrack} + 1} = \frac{\left\lbrack B_{T} \right\rbrack}{\lbrack B\rbrack}}} & {{eq}.\mspace{14mu} 1} \\{{\lbrack B\rbrack = {{\frac{\left\lbrack B_{T} \right\rbrack}{\frac{\left\lbrack {B \cdot S} \right\rbrack}{\lbrack B\rbrack} + 1}\left\lbrack {B \cdot S} \right\rbrack} = {\frac{\left\lbrack {B \cdot S} \right\rbrack}{\lbrack B\rbrack}\lbrack B\rbrack}}},{{{where}\mspace{14mu}\lbrack B\rbrack}\mspace{14mu} {is}\mspace{14mu} {calculated}\mspace{14mu} {from}\mspace{14mu} {{eq}.\mspace{14mu} 1}}} & {{eq}.\mspace{14mu} 2} \\{{{\left\lbrack {B \cdot S} \right\rbrack + \lbrack S\rbrack} = {{\left\lbrack S_{T} \right\rbrack \lbrack S\rbrack} = {\left\lbrack S_{T} \right\rbrack - \left\lbrack {B \cdot S} \right\rbrack}}},{{where}\mspace{14mu} - {\left\lbrack {B \cdot S} \right\rbrack \mspace{14mu} {is}\mspace{14mu} {calculated}\mspace{14mu} {from}\mspace{14mu} {eq}\mspace{14mu} 2}}} & {{eq}.\mspace{14mu} 3}\end{matrix}$

Each value of K_(a) arose from at least two independent experiments withfreshly prepared solutions, and each experiment consisted of a titrationwith 6-9 different concentrations.

All NMR spectra were analyzed with Topspin 3.0 software from Bruker AXS.NMR experiments were done in a 0.10 M NaH₂PO₄ buffer, pH 7.4, containingD₂O (2% v/v). ¹H NMR experiments consisted of the first increment of a2D NOSY with gradients for improved water suppression.

A. Representative Procedure for Making a Phenylboronic Acid SolutionNaH₂PO₄ (3.0 g, 25 mmol) and PBA (phenylboronic acid, 458 mg, 3.75 mmol)were dissolved in distilled, deionized water in a volumetric flask (200mL H₂O, 5 mL D₂O). The pH was adjusted carefully to 7.4 using 10 M NaOH,and additional water was added for a final volume of 250 mL. Finalsolutions were 15 mM in the boronic acid (PBA=solution 1;benzoboroxole=solution 2) in 0.10 M sodium phosphate monobasic buffer,pH 7.4, containing D₂O (2% v/v).

Determination of the Value of K_(a) for PBA and Fructose

To a 25-mL volumetric flask, D-fructose (674 mg, 3.75 mmol) and 20 mL ofsolution 1 was added. The solution was adjusted carefully to pH 7.4 bythe addition of 10 M NaOH. (The volume of added NaOH was used in thecalculation of the boronic acid concentration.) The volume was thenincreased to 25 mL by adding solution 1. This procedure resulted in a pH7.4 solution of PBA (15 mM), D-fructose (150 mM), NaH₂PO₄ (0.10 M), andD₂O (2% v/v) (solution A). Mixing various volumes of solution 1 andsolution A generated fructose concentrations in the range of 4-14 mM.The [B·S]/[B] ratio was determined for every concentration as follows.

Representative Procedure for Determining the Chemical Shifts of ArylProtons in Bound and Free Boronic Acids

A ¹H-NMR spectra of solution 1 (FIG. 13A) and solution A (FIG. 13B) wereacquired. The two spectra were overlaid to determine which peaksbelonged to the bound boronic acid and free boronic acid (FIG. 13C).This analysis was used to interpret the spectra from the titrations withsugars (FIG. 13D).

Determination of the Value of K_(a) for Benzoboroxole and Fructose

To a 25-mL volumetric flask, D-fructose (674 mg, 3.75 mmol) and 20 mL ofsolution 2 was added. The solution was adjusted carefully to pH 7.4 bythe addition of 10 M NaOH. (The volume of added NaOH was used in thecalculation of the boronic acid concentration.) The volume was thenincreased to 25 mL by adding solution 2. This procedure resulted in a pH7.4 solution of benzoboroxazole (15 mM), D-fructose (150 mM), NaH₂PO₄(0.10 M), and D₂O (2% v/v) (solution B). Mixing various volumes ofsolution 2 and solution B generated fructose concentrations in the rangeof 4-14 mM. The [B·S]/[B] ratio was determined as depicted in FIG. 14. Avalue for K_(a) was calculated for every concentration as describedabove.

Determination of the Value of K_(a) for PBA and Glucose

To a 25-mL volumetric flask, D-glucose (2.25 g, 12.5 mmol) and 20 mL ofsolution 1 was added. The solution was adjusted carefully to pH 7.4 bythe addition of 10 M NaOH. (The volume of added NaOH was used in thecalculation of the boronic acid concentration.) The volume was thenincreased to 25 mL by adding solution 1. This procedure resulted in pH7.4 solution of PBA (15 mM), D-glucose (500 mM), 0.1 M NaH₂PO₄ (0.10 M),and D₂O (2% v/v) (solution C). Mixing various volumes of solution 1 andsolution C generated glucose concentrations in the range of 20-70 mM.The [B·S]/[B] ratio was determined as depicted in FIG. 15. A value forK_(a) was calculated for every concentration as described above.

Determination of the Value of K_(a) for Benzoboroxole and Glucose

To a 25-mL volumetric flask, D-glucose (2.25 g, 12.5 mmol) and 20 mL ofsolution 2 was added. The solution was adjusted carefully to pH 7.4 bythe addition of 10 M NaOH. (The volume of added NaOH was used in thecalculation of the boronic acid concentration.) The volume was thenincreased to 25 mL by adding solution 2. This procedure resulted in a pH7.4 solution of benzoboroxazole (15 mM), D-glucose (500 mM), NaH₂PO₄(0.10 M), and D₂O (2% v/v) (solution D). Mixing various volumes ofsolution 2 and solution D generated glucose concentrations in the rangeof 20-70 mM. The [B·S]/[B] ratio was determined as depicted in FIG. 16.Note the broadening of the aryl protons, which had been reported for NMRspectra of boronic acids in the presence of pyranose sugars. (M. Bérubé,M. Dowlut, D. G. Hall, J. Org. Chem. 2008, 73, 6471-6479.) A value forK_(a) was calculated for every concentration as described above.

Determination of the Value of K_(a) for PBA and Neu5Ac

To a 10-mL volumetric flask, Neu5Ac (1.53 g, 5.0 mmol) and 20 mL ofsolution 1 was added. The solution was adjusted carefully to pH 7.4 bythe addition of 10 M NaOH. (The volume of added NaOH was used in thecalculation of the boronic acid concentration.) The volume was thenincreased to 25 mL by adding solution 1. This procedure resulted in a pH7.4 solution of PBA (14.2 mM), Neu5Ac (500 mM), NaH₂PO₄ (0.10 M), andD₂O (2% v/v) (solution E). Mixing various volumes of solution 1 andsolution E, generated Neu5Ac concentrations in the range of 7-65 mM. The[B·S]/[B] ratio was determined as depicted in FIG. 17. A value for K_(a)was calculated for every concentration as described above.

Determination of the Value of K_(a) for Benzoboroxole and Neu5Ac

To a 10-mL volumetric flask, Neu5Ac (1.56 g, 5.0 mM) and 20 mL ofsolution 2 was added. The solution was adjusted carefully to pH 7.4 bythe addition of 10 M NaOH. (The volume of added NaOH was used in thecalculation of the boronic acid concentration.) The volume was thenincreased to 25 mL by adding solution 2. This procedure resulted in a pH7.4 solution of benzoboroxazole (14.2 mM), Neu5Ac (500 mM), NaH₂PO₄(0.10 M), and D₂O (2% v/v) (solution F). Mixing various volumes ofsolution 2 and solution F generated Neu5Ac concentrations in the rangeof 7-65 mM. The K_(a) value was calculated for every concentration aspreviously described. The [B·S]/[B] ratio was determined as illustratedin FIGS. 18 and 19. Apparent in the ¹H NMR spectrum of Neu5Ac (FIG. 18)is a small peak that overlapped with the aromatic regions of the boronicacids. This peak was subtracted out of all NMR spectra used to evaluatethe interaction of benzoboroxazole and Neu5Ac. Unlike similarexperiments with fructose and glucose, the single isolated proton of thecomplexed species (7.33 ppm) was too broad to integrate accurately, andthe entire region was used instead. A value for K_(a) was calculated forevery concentration as described above.

Table 1 summaries K_(a) data measured as described above with comparisonto literature values. Measured K_(a) values are in agreement with thosemeasured by others employing alternative assays. Both PBA andbenzoboroxole were found to have greater affinity for Neu5Ac than forglucose. Benzoboroxole was found to have greater affinity than PBA foreach saccharide measured.

TABLE 1 Values of K_(a) (M⁻¹) for boronic acids and saccharides BoronicD- D- Acid fructose glucose Neu5Ac Method Ref. PBA 128 ± 20  5 ± 1 13 ±1 ¹H NMR in H₂O containing This D₂O (2% v/v) [a] work 160 4.6 21Competition with alizarin red [b] S [a]  79 0 — ¹H NMR in D₂O (100% v/v)[a] [c] — — 11.6 ± 1.9 ¹¹B NMR in H₂O/D₂O/MeOH [d] mixture Benzo- 336 ±43 28 ± 4 43 ± 5 ¹H NMR in H₂O containing This boroxole D₂O (2% v/v) [a]work 606 17 — ¹H NMR in D₂O (100% v/v) [a] [c] — 31 — Competition withalizarin red S [a] [e] [a] Values were determined in 0.10M sodiumphosphate buffer, pH 7.4; [b] G. Springsteen, B. Wang, Tetrahedron 2002,58, 5291-5300; [c] M. Dowlut, G. Dennis, J. Am. Chem. Soc. 2006, 128,4226-4227; [d] K. Djanashvili, L. Frullano, J. A. Peters, Chem. Eur. J.2005, 11, 4010-4018; [e] M. Bérubé, M. Dowlut, D. G. Hall, J. Org. Chem.2008, 73, 6471-6479.

Example 3 Conjugation of 5-amino-2-hydroxymethylphenylboronic acid toRNase A Generating Benzoboroxole-Boronated RNase A

To display benzoboroxole moieties on RNase A,5-amino-2-hydroxymethyl-phenylboronic acid (1) was conjugated to proteincarboxyl groups by condensation using a carbodiimide:

Also see Scheme 1 (FIG. 1A). As noted below of the 11 carboxyl groups ofRNase A, on average 5.7±1.6 were condensed with benzoboroxole asdetermined by mass spectrometry.

5-Amino-2-hydroxymethylphenylboronic acid (1, 320 mg, 1.70 mmol) wasadded to 30 mL of distilled, deionized H₂O, and the resulting solutionwas adjusted to pH 5.0 with NaOH. To this solution was added RNase A(200 mg, 15 mmol), followed by EDC (640 mg, 3.30 mmol), and the pH wasadjusted again to 5.0 with NaOH. The reaction mixture was incubated atambient temperature overnight on a nutating mixer by BD (Franklin Lakes,N.J.). Additional EDC (360 mg, 1.9 mmol) was added, and the solution wasincubated at the same conditions for 3.5 h (24 h total). The solutionwas then subjected to centrifugation (5 min at 1000 rpm, and 5 min at5000 rpm) to remove insoluble boronic acid, and dialyzed (3500 molecularweight cutoff) against distilled, deionized H₂O for 3 d at 4° C., withdaily water exchanges. The solution was then passed through a 0.45-μmfilter and loaded onto a 5-mL HiTrap Heparin HP column. To prepare ahigh-salt buffer, NaCl (58.4 g, 1.00 mol) was added to 100 mL of a 10×stock solution of PBS. This solution was diluted with distilled,deionized H₂O to a final volume of 1 L, and adjusted to pH 7.4, making abuffer of PBS plus an additional 1 M NaCl. The column was washed with 75mL of PBS buffer, and protein was eluted with a linear gradient of 225mL of additional NaCl (0.0-1.0 M) in PBS buffer. Fractions werecollected, pooled, concentrated, stored at 4° C., and analyzed byMALDI-TOF mass spectrometry.

The mass spectrum between 13-16 kDa was fitted to a Gaussian curve withGraphPad Prism version 5.02 software to determine the average mass (seeFIG. 20). Specifically, the mean molecular mass determined for theconjugated RNase A (14,664 Da) was subtracted from the observedmolecular mass of unmodified RNase A (13,682 Da) to give 982 Da. Thisvalue was divided by the molecular mass of5-amino-2-hydroxymethylphenylboronic acid corrected for the loss ofwater during conjugation (185.42 Da−18.02 Da=167.40 Da) to give 5.9±1.6phenylboronic acids conjugated to RNase A, where SD=1.6 arises from theSD of the Gaussian fit, 265.2 Da, divided by 167.42 Da.

Example 4 Conjugation of 3-amino-phenylboronic acid to RNase AGenerating PBA-Boronated RNase A

PBA boronatedRNase A was synthesized similarly tobenzoboroxole-boronated RNase A as described in Example 3.

To 30 mL of double deionized H₂O was added 3-aminophenylboronic acid (2,118 mg, 0.9 mmol) and the pH of the solution was adjusted to 5.0 withNaOH. To this solution RNase A (100 mg, 7 μmol) was added, followed byEDC (320 mg, 1.7 mmol), and the pH was re-adjusted to 5.0 with NaOH.Reaction was incubated at ambient temperature overnight on a nutatingmixer. The solution was then centrifuged briefly (5 min at 1 k rpm, 5min at 5 k rpm) to remove insoluble boronic acid and dialyzed (3500MWCO) against double deionized H₂O for 3 d at 4° C., with daily waterexchanges. Proteins were loaded onto a 1 mL HiTrap Heparin HP column. Toprepare a high-salt buffer, NaCl (58.4 g, 1.00 mol) was added to 100 mLof a 10× stock solution of PBS. This solution was diluted withdistilled, deionized H₂O to a final volume of 1 L, and adjusted to pH7.4, making a buffer of PBS plus an additional 1 M NaCl. The column waswashed with 30 mL of PBS buffer, and protein was eluted with a lineargradient of 90 mL of additional NaCl (0.0-1.0 M) in PBS buffer.Fractions were collected, pooled, and analyzed by MALDI-TOF massspectrometry.

The mass spectrum between 13-16 kDa was fitted to a Gaussian curve withGraphPad Prism version 5.02 software to determine the average mass asdescribed above. Unmodified RNase A control: measured mass 13,657±285.16Da, expected mass 13,682 Da, therefore, the correctionfactor=13,682−13,657=25 Da. PBA RNase A: measured mass 14,285±269.7 Da;adding correction factor=gives correct mass of 14,285+25=14,310 Da.Subtracting unmodified RNase mass from corrected mass of conjugate gives14,310−13,682=628 Da. Each PBA conjugation is 118.79 Da, so on average5.3±2.3 PBA are conjugated to RNase A.

Example 4 Preparation of Inactivated, Benzoboroxole-Boronated RNase A

RNase A was inactivated by treatment with 2-bromoacetic acid. RNase A(38 mg, 2.8 mol) was dissolved in 575.5 μL of 0.10 M sodium acetatebuffer, pH 4.9. In a separate solution, 2-bromoacetic acid (123 mg, 883mol) was dissolved in 9.2 mL of 0.10 M sodium acetate buffer. Theresulting solutions were adjusted to pH 5.2. An aliquot (288 μL) of the2-bromoacetic acid solution was added to the RNase A solution togenerate a final concentration of 32 mM 2-bromoacetic acid and 3.2 mMRNase A. The reaction mixture was incubated at ambient temperature for24 h on a nutating mixer, after which the reaction was dialyzedovernight against distilled, deionized H₂O.

The inactivated RNase A was then loaded onto a Mono S HR 16/10 cationexchange FPLC column from Pharmacia. The column was washed with a 40-mLlinear gradient of NaCl (0.00-0.05 M) in 10 mM sodium phosphate buffer,pH 6.0, and eluted with a 603-mL linear gradient of NaCl (0.05-0.40 M)in 10 mM sodium phosphate buffer, pH 6.0. Fractions were collected,pooled, and dialyzed overnight at 4° C. against 50 mM sodium acetatebuffer, pH 5. Inactivated RNase A was then loaded onto a 5-mL HiTrapHeparin HP column. The column was washed with 10 mL of 50 mM sodiumacetate buffer, pH 5.0, and eluted with a 200-mL linear gradient of NaCl(0.0-0.4 M) in 50 mM sodium acetate buffer, pH 5.0. Fractions werecollected, and analyzed by MALDI-TOF mass spectrometry. Fractions withmolecular mass greater than that of unmodified RNase A were pooled anddialyzed extensively with distilled, deionized H₂O at 4° C.

5-Amino-2-hydroxymethylphenylboronic acid (1) was then conjugated to theinactivated RNase A as described above. Briefly, to 0.5 mL of chemicallyinactivated RNase A (6 mg, 400 nmol) was added5-amino-2-hydroxymethylphenylboronic acid (10 mg, 50 μmol), and adjustedto pH 5. EDC was then added (19 mg, 100 μmol), and the resultingsolution was adjusted to pH 5. The reaction mixture was incubated atambient temperature for 20.5 h on a nutating mixer before addingadditional EDC (11 mg, 56 μmol), and then incubated for an additional3.5 h. Inactivated, benzoboroxole-boronated RNase A was dialyzed againstdistilled, deionized H₂O and purified on a 1-mL HiTrap Heparin HP columnas described for benzoboroxole-boronated RNase A.

Example 5 Preparation of BODIPY FL-Labeled Ribonucleases

A. Unmodified and benzoboroxole-conjugated RNase A were labeled withBODIPY FL. An aliquot (3.83 mL) of a solution of ribonuclease (120 μM)was adjusted to pH 8.3. BODIPY FL STP ester (5 mg; 9 μmol) was dissolvedin 0.5 mL of DMF. To the solution of ribonuclease was added 125 μL ofthe BODIPY FL STP ester solution. The reaction mixture was incubated atambient temperature on a nutating mixer for 4-6 h, and then incubated at4° C. on a nutating mixer overnight. Labeled ribonuclease was loadedonto a 1-mL HiTrap Heparin HP column. The column was washed with 30 mLof mM sodium phosphate buffer, pH 6.0. The protein was eluted with a60-mL linear gradient of NaCl (0.0-1.0 M) and pH (6.0-7.4) in 10 mMsodium phosphate buffer, pH 7.4. Fractions were collected, pooled,concentrated, and analyzed by SDS-PAGE and MALDI-TOF mass spectrometry.

Labeled ribonucleases were dissolved in at least a 10× volume of DPBS,passed through a 0.45-μm syringe filter from Whatman (Piscataway, N.J.),and re-concentrated before being used in assays. In this manner, theproteins were dissolved in solution that was largely DPBS.

Concentrations of proteins were determined by UV spectroscopy using theextinction coefficient of RNase A at 278 nm (ε=0.72 (mg·mL⁻¹)⁻¹·cm⁻¹).[M. Sela, C. B. Anfinsen, W. F. Harrington, Biochim. Biophys. Acta 1957,26, 502-512.] The absorbance of benzoboroxole was found to benegligible, contributing <5% to the A_(278 nm) of the boronatedribonuclease. The concentration of labeled ribonucleases was correctedfor fluorophore absorbance by using the manufacturer's protocol(http://tools.invitrogen.com/content/sfs/manuals/mp00143.pdf). Percentlabeling was determined by UV spectrometry at 504 nm using theextinction coefficient of BODIPY FL (ε=68,000 M⁻¹·cm⁻¹) as per themanufacturer's protocol. The boronated-RNase A conjugate achieved 30%labeling, whereas RNase A achieved 89% labeling.

B. Unmodified and PBA-boronated RNase A were labeled with BODIPY FLsimilar to unmodified and benzoboroxole-boronated RNase A describedabove. An aliquot (20 mL) of a solution of a fraction of PBA-boronatedRNase A (fraction 3) and unmodified ribonuclease (45 μM) was adjusted topH 8.8. BODIPY-FL STP ester (5 mg; 9 μmol) was dissolved in 0.5 mL ofDMF. To the solutions of ribonuclease was added 250 μL of the BODIPY FLSTP ester solution. PBS was added to the solution for a total volume of50 mL. The reaction mixture was incubated at ambient temperature on anutating mixer for approximately 2 h, and then dialyzed against PBS at4° C. overnight. Labeled ribonuclease was loaded onto a 1-mL HiTrapHeparin HP column. The column was washed with 30 mL of 10 mM sodiumphosphate buffer, pH 7.4. The protein was eluted with a 60-mL lineargradient of 0.0-1.0 M NaCl in 10 mM sodium phosphate buffer, pH 7.4.Fractions were collected, pooled, concentrated, and analyzed by SDS-PAGEand MALDI-TOF mass spectrometry.

Concentrations of proteins were determined by UV spectrometry using theextinction coefficient of RNase A at 278 nm (ε=0.72 (mg·mL−1)−1 cm−1)(Sela 1957). The concentration of labeled ribonucleases was correctedfor fluorophore absorbance by using the manufacturer protocol(http://tools.invitrogen.com/content/sfs/mauals/mp00143.pdf). Percentlabeling was determined by UV spectrometry at 504 nm using theextinction coefficient of BODIPY FL (ε=68,000 M−1·cm−1) as per themanufacturer's protocol. Boronated RNase A achieved 30% labeling andRNase A achieved 59%.

Example 6 Heparin-Affinity Assays

To assess the affinity of boronated proteins for oligosaccharides theretention of boronated proteins compared to unmodified protein can bemeasured on a heparin column.

For example, the affinity of unmodified and benzoboroxole-boronatedRNase A for heparin was assessed by retention on a 1.0-mL HiTrap HeparinHP column (GE Healthcare, Piscataway, N.J.). Unmodified andbenzoboroxole-boronated RNase A were mixed in a 1:1 ratio (2.0 mg each)in DPBS, and the resulting solution was loaded onto the column. Thecolumn was washed with 5 mL of PBS, followed by elution with 45 mL of alinear gradient of NaCl (0.0-1.0 M) in PBS. Elution was monitored byabsorbance at 280 nm, and eluted proteins were identified by massspectrometry. FIG. 2 illustrates an elution profile of a mixture ofunmodified RNase A and benzoboroxole-boronated RNase A from a column ofimmobilized heparin (solid lines). A small amount of unmodified RNase Awas apparent in peak B (FIG. 2). It is currently believed thatbenzoboroxole-boronated RNase A was able to complex to a small amount ofunmodified RNase A and extend its elution time. The same assay was thenrepeated with 100 mM fructose in both buffers (FIG. 2, dashed line). Tomake fructose-supplemented buffers, fructose (18 g, 100 mmol) was addedto 100 mL of a 10× stock solution of PBS, either no additional NaCl orNaCl (58.4 g, 1.00 mol) was added, and both buffers were diluted to afinal volume of 1 L and adjusted to pH 7.4.

As shown in FIG. 2, benzoboroxole-boronated RNase A was indeed retainedlonger on the column. Addition of fructose in the buffer employed wasshown to compete with immobilized heparin for boron complexationdiminishing retention of benzoboroxole-boronated RNase A and indicatingthat prolonged retention of benzoboroxole-boronated RNase A was due toboron-saccharide complexation.

Example 7 Assessing the Affinity of Benzoboroxole-Boronated-RNase A forGD3 Ganglioside-Labeled Liposomes Employing Fluorescence PolarizationAssays

To evaluate the enhanced affinity of benzoboroxole-boronated RNase A foroligosaccharides, its affinity for ganglioside GD3 within a1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposome was measured.This ganglioside has two sialic acid residues and is overexpressed onthe surface of cancer cells. (F. Malisan, R. Testi, IUBMB Life 2005, 57,477-482.) By using fluorescence polarization to analyze binding, it wasdemonstrated that boronation increased the affinity of the protein forthe ganglioside, an effect that was abrogated by fructose (FIG. 3). TheK_(d) value of benzoboroxole-boronated protein for GD3 gangliosideliposomes was (54±11) M. This affinity is ˜430 fold greater than thatfor the binding of a single benzoboroxole to Neu5Ac (Table 1),consistent with a multivalent interaction between thebenzoboroxole-boronated protein and the ganglioside.

Liposomes were formed by transferring DOPC (dissolved in chloroformsolution) and GD3 gangliosides (dissolved in 63:35:5chloroform/methanol/water) to glass tubes and drying them under Ar(g)and then under vacuum. Lipids were re-suspended in 25 mM HEPES buffer,pH 7.0, containing NaCl (75 mM). The solution of lipids was mixed byvortexing for 2 min, and incubated at 37° C. for 1 h. For DOPCliposomes, DOPC was resuspended at a concentration of 5 mM. For GD3ganglioside-labeled liposomes, DOPC and GD3 gangliosides were mixed at 3mM and 2 mM concentrations, respectively. Large unilammelar vesicleswere formed by extrusion through a 0.1-1 μm polycarbonate filter fromWhatman (GE Healthcare, Piscataway, N.J.). This process produces apopulation of vesicles of near uniform size (100-150 nm diameter asmeasured by dynamic light scattering). A portion of the DOPC lipidsbefore extrusion were aliquoted as a control.

Fluorescence polarization assays were performed using 50 nM BODIPYFL-labeled unmodified and benzoboroxole-boronated RNase A in black NBS96-well plates (Corning Costar, Lowell, Mass.). These ribonucleases wereincubated with DOPC liposomes (625 μM total lipid) or GD3ganglioside-labeled liposomes (375 μM DOPC, 250 μM GD3 ganglioside=625μM total lipid) in 25 mM HEPES buffer, pH 7.0, containing NaCl (75 mM)in the absence or presence of fructose (10 mM). In control wells,ribonucleases were incubated with non-extruded DOPC lipids. Fluorescencepolarization at 470/535 nm with a G-factor of 1.257 was recorded aftershaking the plate briefly and incubating at ambient temperature for 1 h.Control well polarization was subtracted from experimental wellpolarization for each ribonuclease. The assay was performed intriplicate.

The affinity of benzoboroxole-boronated RNase A for GD3ganglioside-labeled liposomes was assessed by using serially dilutedliposomes. GD3 ganglioside-labeled liposomes were serially diluted in 25mM HEPES, pH 7.0, containing 75 mM with dilutions of 62.5 nM-1250 μMtotal lipid. Because the composition of these liposomes was 3:2 DOPC/GD3ganglioside, this dilution resulted in a solution containing D3ganglioside at 25 nM-500 μM. Control DOPC liposomes (with no GD3ganglioside) were likewise diluted in the same buffer, producingsolutions of 62.5 nM-1250 μM total lipid. Liposomes were then incubatedwith 50 nM BODIPY FL-labeled benzoboroxole-boronated RNase A in the samebuffer in a black NBS 96-well plate. Fluorescence polarization wasrecorded after shaking the plate briefly and incubating at ambienttemperature for 35 min. Fluorescence polarization from GD3ganglioside-labeled liposomes was subtracted from that from DOPC-onlyliposomes, thereby correcting for binding to DOPC and for changes insolution viscosity. The assay was performed in duplicate. The fractionof labeled ribonuclease bound for each sample well was calculated bydividing its polarization from the polarization of ribonucleasesincubated with the highest concentration of GD3 ganglioside (set at 100%bound). The value of K_(d) was calculated by plotting the fraction boundagainst the concentration of GD3 ganglioside (see FIG. 21) and fittingthe data to a binding isotherm as described in M. H. Roehrl, J. Y. Wang,G. Wagner, Biochemistry 2004, 43, 16056-16066 to give Kd=(54±11) μM.

Example 8 Cellular Internalization RNase Conjugates

To quantify cellular internalization, Fluorophore-labeled protein andflow cytometry were used to quantify cellular internalization ofboronated protein. To determine concurrently if the pendant boronateselicited selectivity for cells with higher quantities of cell-surfacesialic acid, a line of Chinese hamster ovary cells (Lec-2) was used.These cells have lower levels of sialic acid in their glycocalyx, thantheir progenitor line (Pro 5). (S. L. Deutscher, N. Nuwayhid, P.Stanley, E. I. Briles, C. B. Hirschberg, Cell 1984, 39, 295-299). Asshown in FIG. 4A boronation of RNase A with benzoboroxole increased itscellular uptake by 4- to 5-fold in both cell lines tested. As shown inFIG. 4C boronation of RNase A with PBA increased its cellular uptake by2.5-3 fold in both cell lines tested. In both cases, this enhancementwas eliminated by addition of fructose.

Cell-surface sialic acid-content (comparing Pro-5 to Lec-2 cells) wasnot observed to affect uptake significantly. This result is consistentwith the modest increase observed in the K_(a) value for benzoboroxolewith sialic acid versus glucose (Table 1).

Confocal microscopy of boronated protein (benzoboroxole-boronated RNase)demonstrated punctate staining (FIG. 4B), which is consistent withuptake by endocytosis following complexation with cell-surfacesaccharides.

A. Cell Culture

Cell lines were obtained from American Type Culture Collection(Manassas, Va.) and were maintained according to the recommendedprocedures. Cells were grown in a cell culture incubator at 37° C. underCO₂ (5% v/v) in flat-bottomed culture flasks. Cell medium wassupplemented with GIBCO fetal bovine serum (FBS) (10% v/v), penicillin(100 units/mL), and streptomycin (100 μg/mL) in the appropriate cellularmedium as follows: Pro-5, MEM α+ribonucleosides+deoxyribonucleosides;Lec-2, MEM α-ribonucleosides-deoxyribonucleosides; and K562, RPMI 1640.Cells were counted by hemocytometry for dispensing into 12-well plates(Corning Costar, Lowell, Mass.) or 8-well chambered coverglass slides(Nuc Lab-Tek II, Thermo Scientific).

B. Flow Cytometry Assays

BODIPY-FL was excited with a 488 nm solid-state laser and the emissionwas collected with a 530/30 bandpass filter. To collect the mostreproducible data, for every flow cytometry experiment, the sensitivity(voltage) of the photomultiplier tube was set for all data collectionsusing mid-range Rainbow beads from Spherotech (Lake Forest, Ill.) to apredetermined fluorescence target value. At least 10,000 cellular eventswere acquired for each sample. Data were analyzed using FlowJo 8.1.3(Treestar, Ashland, Oreg.).

The day prior to an experiment, Pro-5 and Lec-2 cells were plated in12-well plates at 1×10⁵ cells/well. The day of the experiment, theappropriate amount of fructose was dissolved into the cellular medium toobtain a final fructose concentration of 250 mM, and the medium waspassed through a 0.45-μm syringe filter from Whatman (Piscataway, N.J.).Non-fructose-containing medium was filtered likewise. Stock solutions offluorescently labeled ribonucleases were diluted into the cell cultureto a final concentration of 5 μM. Ribonucleases were incubated withcells for 4 h. Cells were then rinsed with PBS (2×400 μL), removed fromthe cell culture plate with trypsin (400 μL, 0.05% (1×) with EDTA;Invitrogen, Carlsbad, Calif.), placed in flow cytometry tubes containing80 μL of FBS, and incubated on ice until analyzed by flow cytometry.Final fluorescence values were divided by the percent fluorophorelabeling of the ribonuclease to determine the corrected value offluorescence. Experiments were run twice in triplicate unless otherwiseindicated. Data is illustrated in FIG. 4A and FIG. 4C.

C. Confocal Microscopy

Pro-5 cells were plated on Nunc Lab-tek II 8-well chambered coverglass24 h before use and grown to 80% confluency. Cells were incubated with 5μM BODIPY FL-labeled ribonucleases for 4 h. Cell nuclei were stainedwith Hoechst 33342 (Invitrogen, 2 μg/mL) for the final 15 min ofincubation. Cells were then washed twice with PBS, suspended in PBS, andexamined using a Nikon Eclipse C1 laser scanning confocal microscope.Results are illustrated in FIG. 4B.

Example 9 Assessing Cytosol Delivery

Although flow cytometry can quantify protein internalization into acell, it does not differentiate between proteins in endosomes versusthose in the cytosol. Delivery into the cytosol is important for theefficacy of numerous putative chemotherapeutic agents. Boronated RNase Aretained ribonucleolytic activity (17±2% for benzoboroxole-boronatedRNase A.) The activity loss is believed attributable, at least in part,to the modification of the carboxyl group of aspartic acid 121 onreaction with the boronic acid. Aspartic acid 121 is known to contributeto RNAse A activity (L. W. Schultz, D. J. Quirk, R. T. Raines,Biochemistry 1998, 37, 8886-8898). Boronated RNase A thus has thepotential to be cytotoxic, if it can enter the cytosol.Benzoboroxole-boronated RNase A was found to inhibit the proliferationof a line of human erythroleukemia cells (K-562; FIG. 5). The additionof fructose diminished cytotoxic activity, presumably by decreasingoverall internalization of boronated RNase A. In contrast, chemicallyinactivated, benzoboroxole-boronated RNase A was much less cytotoxic,indicating that ribonucleolytic activity induced toxicity, not thependant boronates. Boronation not only facilitates cellular uptake of anenzyme, but also allows for its delivery to the cytosol and themaintenance of its catalytic activity.

A. Ribonucleolytic Activity Assays

The ribonucleolytic activities of RNase A, boronated RNase A, andboronated inactive RNase A were determined by quantifying their abilityto cleave 6-FAM-dArUdAdA-6-TAMRA, as described in B. R. Kelemen, T. A.Klink, M. A. Behlke, S. R. Eubanks, P. A. Leland, R. T. Raines, NucleicAcids Res. 1999, 27, 3696-3701. Assays were carried out at ambienttemperature in 2 mL of 0.10 M MES-NaOH buffer, pH 6.0, containing NaCl(0.10 M). Fluorescence data were fitted to the equation:k_(cat)/K_(M)=(ΔI/Δt)/(I_(f)−I₀)[E], in which ΔI/Δt is the initialreaction velocity, I₀ is the fluorescence intensity before addition ofribonuclease, I_(f) is the fluorescence intensity after completesubstrate hydrolysis, and [E] is the total ribonuclease concentration.The assay was performed in triplicate.

B. Cell-Proliferation Assays

The effect of unmodified and boronated RNase A on the proliferation ofK-562 cells was assayed as described in P. A. Leland, L. W. Schultz, B.M. Kim, R. T. Raines, Proc. Natl. Acad. Sci. USA 1998, 95, 10407-10412.For assays, 5 μL of a solution of the ribonuclease or PBS (control) wasadded to 95 μL of cells (5.0×10⁴ cells/mL). For co-treatment assays withfructose, ribonucleases were first serially diluted at 2×concentration,followed by addition of an equal volume of 2 M fructose in PBS to eachribonuclease dilution, resulting in a 1×ribonuclease dilution as beforebut now containing 1 M fructose. Then, 5 μL of each dilution was addedto cells as above, including a control of PBS containing 1 M fructose.Because 5 μL of samples were added to 95 μL of cells, the finalconcentration of fructose in each well was 50 mM. After a 44-hincubation, K-562 cells were treated with [methyl-³H]thymidine for 4 h,and the incorporation of radioactive thymidine into cellular DNA wasquantitated by liquid scintillation counting. The results are shown asthe percentage of [methyl-³H]thymidine incorporated relative to controlcells treated with PBS in FIG. 5. Data are the average of threemeasurements for each concentration, and the entire experiment wasrepeated in triplicate. Values for IC50 were calculated by fitting thecurves by nonlinear regression to the equation: y=100%/(1+10^((log(IC)⁵⁰ ^()−log[ribonuclease])h)), in which y is the total DNA synthesisfollowing the [methyl-³H]thymidine pulse and h is the slope of thecurve.

Example 10 Boronation of Lysozyme

Boronated lysozyme was synthesized similarly to boronated RNase A asdescribed in Example 3. For the synthesis of the benzoboroxole-boronatedlysozyme, 5-amino-2-hydroxymethylphenylboronic acid (1, 160 mg, 0.9mmol) was added to 20 mL of double deionized H₂O and the pH of thesolutions were adjusted to 5.0 with NaOH. To the solution was addedlysozyme (from chicken egg white, 20 mg, 1.4 mol), followed by EDC (320mg, 1.7 mmol), and the pH was re-adjusted to 5 with NaOH. PBA-boronatedlysozyme was analogously synthesized employing 3-aminophenyboronic acid(2, 160 mg, 1.2 mmol). Reactions were incubated at ambient temperatureovernight on a nutating mixer. The reaction solutions were thencentrifuged briefly (5 min at 1 k rpm, 5 min at 5 k rpm) to removeinsoluble boronic acid and dialyzed (3500 MWCO) against double deionizedH₂O for 3 d at 4° C., with daily water exchanges.

Proteins were loaded onto a 1 mL HiTrap Heparin HP column. The columnwas washed with 20 mL of 10 mM sodium phosphate buffer, pH 7.4. Proteinwas eluted with a 10 mL linear gradient of 0.0-1.5 M NaCl in 10 mMsodium phosphate buffer, pH 7.4. Fractions were collected, pooled, andanalyzed by MALDI-TOF mass spectrometry. The mass spectrum between 13-16kDa was fitted to a Gaussian curve with GraphPad Prism version 5.02software to determine the average mass and calculate the number ofboronates conjugated/lysozyme.

Lysozyme control: 14,260±52 Da, expected 14,307 Da (from Sigma Aldrichwebsite of similar protein with reference to Canfield, R. E., J. Biol.Chem., 238, 2698-2707 (1963)). Therefore, the correction factor=14,307Da−14,260 Da=47 Da. For PBA-boronated lysozyme, the measured mass was14,568±199 Da+47 Da (correction factor)=14,615±199 Da. Mass due toconjugated PBA is then 308 Da and the mass of each PBA is 118.79 Da. Onaverage, 2.6±1.7 PBA are conjugated to the lysozyme. Forbenzoboroxole-conjugated lysozyme a similar calculation, using measuredcorrected mass for benzoboroxole-conjugated lysozyme of 14,700±187 Da,and mass/benzoboroxole conjugation of 167.4 Da, gives on average 2.3±1.1benzoboroxole conjugated per lysozyme.

BODIPY FL-labeled lysozyme and BODIPY FL-labeled boronated lysozyme weresynthesized similarly to BODIPY FL-labeled RNase A and boronated RNase Aas described in Example 5. Lysozyme, PBA-boronated lysozyme, andbenzoboroxole-boronated lysozyme were diluted with 0.1 M sodiumbicarbonate buffer (pH 8.3) to 3 mg/mL in 550 μL total (1.65 mg, 115nmol). To this was added 25 μL of 5 mg/mL BODIPY FL, STP ester dissolvedin 0.1 M sodium bicarbonate buffer (pH 8.3) (0.125 mg, 231 nmol, 2×). Anadditional 575 μL of 0.1M sodium bicarbonate buffer (pH 8.3) was added.The reaction was incubated at ambient temperature for about 2 h on anutating mixer. Labeled proteins were then loaded onto a 1 mL HiTrapHeparin HP column. The column was washed with 20 mL of 10 mM sodiumphosphate buffer (pH 7.4). Protein was eluted with a 40 mL lineargradient of 0.0-1.5 M NaCl in 10 mM sodium phosphate buffer (pH 7.4).Fractions were collected, and those with the most fluorescence at501/522 nm were pooled and concentrated. Note that in a SDS-PAGE gel,there was a small molecular weight fluorescent contaminant in theBODIPY-labeled unmodified lysozyme control, but that >50% of the labeledproduct was at the correct molecular weight.

Concentrations of lysozyme and lysozyme conjugates were estimated by UVspectrometry at 280 nm using the extinction coefficient of lysozyme at281.5 nm (c=26.4 (1% w/v)⁻¹ cm⁻¹) (Aune, K. C. and Tanford, C.Thermodynamics of the Denaturation of Lysozyme by GuanidineHydrochloride. I. Dependence on pH at 25° C. Biochemistry 1969, 8,4579-4585). The concentration of labeled lysozymes were corrected forBODIPY FL absorbance following the manufacturer's protocol (which can befound at http://tools.invitrogen.com/content/sfs/manuals/mp00143.pdf)using the following equation: Absorbanceprotein=A_(280 nm)−A_(504 nm)*(CF), where CF=correction factor suppliedby the manufacturer. The percent labeling was determined by UVspectrometry at 504 nm using the extinction coefficient of BODIPY FL(ε=68,000 M-cm⁻¹) as per manufacturer protocol (which can be found athttp://tools.invitrogen.com/content/sfs/manuals/mp00143.pdf) using thefollowing equation: Degree of Labeling=(A_(504 nm)*mw protein)/(proteinmg/mL*ε_(BODIPY)), where molecular weight was assumed as 14.3 kDa forall proteins.

These calculation indicated the degree of labeling for the indicatedspecies:

BODIPY FL lysozyme: 36.7%BODIPY FL PBA-boronated lysozyme: 9.6%BODIPY FL benzoboroxole-boronated lysozyme: 26.0%

Example 11 Cell Internalization of Boronated Lysozyme

As shown in FIG. 6, boronation of lysozyme with benzoboroxole increasedits cellular uptake into HeLa cells by about 4-fold. In contrast,boronation of lysozyme with PBA increased its cellular uptake by about20-fold in HeLa cells. In both cases, this enhancement was decreased byaddition of fructose.

Cell Cultures:

Cell lines were obtained from American Type Culture Collection(Manassas, Va.) and were maintained according to the recommendedguidelines. Cells were grown in a cell culture incubator at 37° C. and5% CO2 in flat-bottomed culture flasks. Cell media was supplemented with10% (v/v) GIBCO fetal bovine serum (FBS), 100 units/mL penicillin, and100 μg/mL streptomycin in the appropriate cellular media for HeLa cells:DMEM, high glucose (Invitrogen, Carlsbad, Calif.). Cells were counted byhemocytometry for dispensing into 12-well plates (Corning Costar,Lowell, Mass.).

Flow Cytometry Assays

BODIPY-FL was excited with a 488 nm solid-state laser and the emissionwas collected with a 530/30 bandpass filter. At least 10,000 cellularevents were acquired for each sample. Data were analyzed using FlowJo8.1.3 (Treestar, Ashland, Oreg.).

The day prior to the experiment, 1× 105 HeLa cells/well were plated in12 well plates. The day of the experiment, the appropriate amount offructose was dissolved into the cellular medium to obtain a final 250 mMfructose concentration, then it was sterile filtered with a 0.45 μmWhatman (Piscataway, N.J.) syringe filter. Non-fructose containingmedium was likewise sterile filtered to maintain consistency between theexperimental groups. Stock solutions of fluorescently labeled proteinswere diluted into the cell culture to a final concentration of 5 μM.proteins were incubated with cells for four hours, then the cells wererinsed twice with PBS, removed from the cell culture plate with trypsin,placed in flow cytometry tubes containing 80 μL of FBS (to ensure cellviability), and incubated on ice until analyzed by flow cytometry. Finalfluorescence values were divided by the percent fluorophore labeling ofthe protein to determine the corrected value of fluorescence.Experiments were run in triplicate (except for PBA-boronated lysozyme,which was in duplicate) and the data is reported as the mean±standarddeviation normalized to the wild type protein uptake.

Example 12 Boronation of a Biotin-Peptide Conjugate and Its Complexationto NeutrAvidin; Cell Internalization of Boronated Avidin

A. Preparation of Boronated Biotin-Peptide Conjugate

The exemplary Biotin-peptide conjugated used was:Biotin-Anx-GEGEGEGEGEGEG-OH, MW: 1531.54 Da, purchased as a customsynthesis from Biomatik (Cambridge, Ontario, Canada), where Anx standsfor ε-aminocaproic acid, G is glycine, and E is glutamic acid.

PBA-boronated biotin-peptide conjugate was prepared similarly toboronated proteins. In 5 mL double deionized water, biotin-peptide (5mg, 3.3 μmol) was added and the pH of the solution was adjusted to 5.0.Then EDC (80 mg, 0.4 mmol) and 3-aminophenylboronic (2, 29.6 mg, 0.2mmol) was added. The solution was left to stir overnight on a nutatingmixer at ambient temperature. After dialysis, PBA-boronatedbiotin-peptide was analyzed by MALDI-TOF.

The biotin-peptide control was measured to have 1528.73 Da major peak,the expected peak in anion mode was 1530 Da, therefore, the correctionfactor was calculated as 1.27 Da. The PBA-boronated biotin-peptideconjugate was measured to have a 2254.20 Da major peak which wascorrected to 2255.47 Da. Each PBA conjugation represents 118.9 Da, so onaverage about 6.1 PBA are conjugated to the biotin-peptide conjugate,noting that 6 glutamates plus the C-terminus are available forconjugation of the phenylboronate.

An analogous method employed 5-amino-2-hydroxymethylphenylboronic acid(1) to prepare benzoboroxole-boronated biotin peptide conjugate.However, the benzoboroxole-boronated biotin peptide crashed out ofsolution and was insoluble. Addition of high concentrations of fructoseto the solution containing the benzoboroxole-boronated biotin peptidesolubilized it.

B. Preparation of Boronated-Avidin

The PBA-boronated biotin-peptide can be used to generate boronatedAvidin. PBA-boronated biotin-peptide conjugate (having on average 6 PBA)was placed into 8 mL of 100 mM fructose in double deionized H₂O (0.625mg/mL, 277 μM). Fluorescein-conjugated NeutrAvidin (5.5 mg), wasdissolved in 1 mL double deionized H₂O (5.5 mg/mL, 91.7 μM). For a totalvolume of 1.8148 mL, an aliquot of 0.1818 mL Fluorescein-conjugatedNeutrAvidin (9 μM final concentration) was added to an aliquot of 1.633mL of peptide (249 μM final concentration, 28×fold excess compared toNeutrAvidin). For control NeutrAvidin, for a total volume of 981.8 μL,an aliquot of 181.8 μL NeutrAvidin (17 μM final concentration) was addedto an aliquot of 800 μL of 0.2 mg/mL biotin (667 μM final concentration,39×fold excess compared to NeutrAvidin). Complexations were incubatedfor 1-2 h at ambient temperature in the dark, then dialyzed overnight in10 mM fructose in double deionized H₂O. Fructose was used throughout tohelp with solubility of the boronates. Note that NeutrAvidin should betetrameric, so 4 peptides should complex, giving about 24 boronicacids/NeutrAvidin.

Concentrations of proteins were determined by UV spectrometry at 494 nmusing the extinction coefficient of fluorescein (ε=68,000 M−1 cm−1)(http://tools.invitrogen.com/content/sfs/manuals/mp00143.pdf) anddividing by 2 as >2 moles fluorescein were conjugated to 1 mole ofNeutrAvidin as per manufacturer protocol(http://www.piercenet.com/coapdfs/CofA-31006-SPECS.pdf). Avidin control(with biotin only) was 414.8 μM fluorescein; ca. 207.4 μM avidin.Avidin-PBA-boronated biotin-peptide complex was 6.78 μM fluorescein; ca.3.39 μM avidin. These results indicate that the boronated biotin-proteincomplex functioned for binding to Avidin.

C. Cell Internalization of Boronated Avidin

Flow cytometry experiments were conducted as described in Example 11with Fluorescein-conjugated NeutrAvidin and BoronatedFluorescein-conjugated NeutrAvidin (boronated by complexation to thePBA-boronated Biotin-peptide conjugate) in HeLa cells. As illustrated inFIG. 7, boronation of Avidin with PBA increased internalization in HeLacells by about 2-fold.

Example 13 Preparation of Phenylboronated Amino Acids for Synthesis ofBoronated Oligopeptides

A. Fmoc-Protected Glutamic Acid Boronated with5-amino-2-hydroxymethyl-phenylboronic acid

Fmoc-L-glutamic acid 1-tert-butyl ester (2.66 g, 6.251 mmol) was addedto a round bottom flask with 1-Hydroxybenzotriazole (HOBT, 1.914 g,12.50 mmol) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU, 3.557 g, 9.377 mmol). The flask was placedunder argon and dry DMF was added (62 mL). N,N-diisopropylethylamine(DIEA, 5.88 mL, 33.78 mmol) was then added at 0° C. and the reaction wasallowed to stir for 10 minutes. Under argon,5-amino-2-hydroxymethyl-phenylboronic acid (2.318 g, 12.0 mmol) wascombined with dry DMF (5 mL) and N,N-diisopropylethylamine (5 mL, 28.72mmol). The solution of amine was added dropwise to the reaction mixtureover an hour. Upon complete addition, the round bottom flask was removedfrom the ice bath and stirred at room temperature for 4 h. The DMF wasthen removed by vacuum. The product was diluted in 200 mL 1 M HCl andextracted with ether (10×200 mL). The organics were concentrated, loadedwith celite, and purified with silica gel chromatography (4% methanol,96% dichloromethane) to yield product (533 mg, 14%). ¹H-NMR (400 MHz;CDCl₃) δ=7.94 (s, 1H), 7.73 (d, J=7.5 Hz, 3H), 7.55 (t, J=8.9 Hz, 2H),7.37 (t, J=7.4 Hz, 2H), 7.29-7.26 (m, 2H), 7.22 (d, J=8.3 Hz, 1H), 5.83(d, J=7.9 Hz, 1H), 4.99 (s, 2H), 4.39 (t, J=6.7 Hz, 2H), 4.29 (m, 1H),4.13 (t, J=6.8 Hz, 1H), 2.42 (m, 2H), 2.30 (m, 1H), 1.99 (m, 1H), 1.45(s, 9H). ¹³C NMR (100 MHz; CDCl₃): δ 176.3, 171.2, 170.9, 157.1, 149.5,143.81, 143.70, 141.4, 137.4, 127.9, 127.2, 125.26, 125.12, 123.3,121.63, 121.54, 120.1, 83.1, 77.2, 71.1, 67.3, 54.0, 47.3, 34.1, 30.1,28.1.

The t-butyl ester protected product was added to a round bottom flaskand 4.0M HCl in dioxane (10 mL, 40 mmol) was added under argon to removethe t-butyl group. The mixture was allowed to stir for 2 hours at rt.The solution was concentrated under reduced pressure and purified byflash chromatography (silica gel, 1:99-10:90, methanol/dichloromethane).¹H-NMR (400 MHz; MeOH) δ=7.78 (m, 7.2 Hz, 3H), 7.65 (m, 3H), 7.36(quintet, J=7.4 Hz, 2H), 7.28 (m, 3H), 4.97 (s, 2H), 4.35-4.27 (m, 2H),4.22 (dd, J=12.5, 6.4 Hz, 1H), 4.12 (t, J=6.7 Hz, 1H), 2.50-2.40 (m,2H), 2.28 (td, J=12.3, 5.7 Hz, 1H), 2.07 (dt, J=15.1, 7.5 Hz, 1H). HRMS(ESI⁻) m/z calculated for (C₂₇H₂₄BN₂O₇)⁻ 498.1708, measured 498.1729.overall yield: 256 mg

B. Fmoc-Protected Glutamic Acid Boronated with 5-aminophenylboronic acid

Fmoc-L-glutamic acid 1-tert-butyl ester (2.61 g, 6.128 mmol) was addedto a round bottom flask with HOBt (1.876 g, 12.26 mmol) and HBTU (3.486g, 9.19 mmol). The flask was placed under argon and dry DMF added (61mL). N,N-diisopropylethylamine (5.67 mL, 32.56 mmol, 0.742 g/mL) wasadded at 0° C. and the reaction was allowed to stir for 10 minutes.Under argon, 3-aminophenylboronic acid pinacol ester (2.685 g, 12.26mmol) was combined with dry DMF (5 mL) and N,N-diisopropylethylamine (5mL, 28.72 mmol, 0.742 g/mL). The solution of amine was added dropwiseover an hour. Upon complete addition, the round bottom was removed fromthe ice bath and allowed to stir at rt for 4 h. The DMF was removed byvacuum. The product was diluted in 200 mL 1 M HCl and extracted withether (3×200 mL). The organics were concentrated, loaded with celite,and purified with silica gel chromatography (40% ethyl acetate, 60%hexanes) to yield product 2.929 g, 80.4%). ¹H NMR (400 MHz; CDCl₃):δ=8.08 (s, 1H), 7.86-7.81 (m, 2H), 7.76 (d, J=7.5, 2H), 7.59 (t, J=8.2,2H), 7.53 (d, J=7.2, 1H), 7.39 (t, J=7.4, 2H), 7.31 (dt, J=7.1, 3.3,2H), 5.61 (d, J=7.7, 1H), 4.42 (t, J=6.6, 2H), 4.29 (t, J=6.8, 1H), 4.20(t, J=6.9, 1H), 2.39 (t, J=6.2, 2H), 2.31 (s, 1H), 1.96 (s, 1H), 1.31(s, 12H), 1.24 (s, 9H). ¹³C NMR (100 MHz; CDCl₃) δ=175.1, 171.1, 170.5,156.9, 143.94, 143.79, 141.5, 137.7, 130.7, 128.6, 127.9, 127.2, 125.9,125.29, 125.20, 123.1, 120.1, 84.0, 83.0, 75.2, 67.3, 47.3, 34.0, 30.0,28.1, 25.0.

The t-butyl ester protected product was added to a round bottom flaskand 4.0M HCl in dioxane (10 mL, 40 mmol) was added under argon. Themixture was allowed to stir for 2 hours at rt. The solution wasconcentrated under reduced pressure and purified by flash chromatography(silica gel, 1:99-10:90, methanol/dichloromethane). ¹H-NMR (400 MHz;CDCl₃) δ=7.93 (s, 1H), 7.77 (m, 4H), 7.57 (d, J=6.4 Hz, 3H), 7.35 (m,7.7 Hz, 4H), 6.04 (d, J=6.9 Hz, 1H), 4.38 (d, J=6.5 Hz, 2H), 4.18 (t,J=6.7 Hz, 1H), 2.65-2.59 (br m, 1H), 2.57-2.51 (br m, 1H), 2.36-2.28 (brm, 1H), 2.14-2.06 (br m, 1H), 1.32 (s, 12H). (KJ-2-104-2). Overallyield: 1.124 g; M-H, Expected: 568.2490, observed: 568.2498.

C. Fmoc-Protected Phenylalanine Boronated with Phenylboronic Acid.

Boc-4-iodo-L-phenylalanine (10 g, 25.56 mmol), bis(pinacolato)diboron(9.737 g, 38.34 mmol), KOAc (12.543 g, 127.8 mmol), PdCl₂(dppf) (1.744g, 2.38 mmol) were added to a round bottom flask. The flask was placedunder argon and dry, degassed DMF added (250 mL). The reaction washeated to 80° C. overnight. The reaction was cooled and filtered througha celite plug and washed with EtOAc. The filtrate was concentrated underreduced pressure and purified by flash chromatography (40%EtOAc/Hexanes, then 100% EtOAc with 1% acetic acid) to yield the crudeproduct (7.4 g). ¹H NMR (400 MHz; CDCl3) δ=7.76 (d, J=7.9, 2H), 7.20 (d,J=7.6, 2H), 4.90 (d, J=6.7, 1H), 4.59 (d, J=7.4, 1H), 3.22 (dd, J=14.4,4.8, 1H), 3.11 (dd, J=15.0, 7.1, 1H), 1.42 (s, 9H), 1.34 (s, 12H).

Boc-4-pinicalborane-L-phenylalanine (3.7 g, 9.456 mmol) was added to around bottom flask with 4.0 M HCl in dioxane (40 ml, 160 mmol HCl) andstirred at room temperature overnight to remove the Boc protectinggroup. The reaction was purged with Ar gas for thirty minutes to removethe HCl gas and concentrated under reduced pressure. ¹H NMR (400 MHz;MeOH) δ=7.76 (d, J=8.0, 2H), 7.32 (d, J=7.9, 2H), 4.27 (dd, J=7.9, 5.6,1H), 3.36 (d, J=5.3, 1H), 3.16 (dd, J=14.6, 8.1, 1H), 1.35 (s, 12H).4-Pinicolborane-L-phenylalanine (4.554 g, 13.80 mmol),N,N-diisopropylethylamine (9.62 mL, 55.21 mmol), and FmocN-hydroxysuccinimide ester (5.133 g, 15.18 mmol) was added to a roundbottom flask. The reaction was put under Ar and anhydrous dioxane (130mL) was added. The reaction was stirred at room temp overnight,concentrated under reduced pressure, and purified by silica flashchromatography (1% MeOH/DCM, then 20% MeOH/DCM v/v). ¹H-NMR (400 MHz;CDCl₃) δ=7.76 (d, J=7.4 Hz, 4H), 7.54 (t, J=7.4 Hz, 2H), 7.40 (t, J=7.5Hz, 2H), 7.30 (m, 2H), 7.18 (d, J=7.3 Hz, 2H), 5.20 (d, J=7.8 Hz, 1H),4.71 (q, J=6.3 Hz, 1H), 4.42 (m, 1H), 4.34 (t, J=8.9 Hz, 1H), 4.20 (t,J=7.2 Hz, 1H), 3.24 (dd, J=13.8, 5.2 Hz, 1H), 3.14 (dd, J=14.2, 5.7 Hz,1H), 1.34 (s, 12H). ¹³C NMR (126 MHz; CDCl3): δ=174.9, 155.9, 143.90,143.77, 141.4, 138.9, 135.3, 128.9, 127.9, 127.2, 125.26, 125.21, 120.1,84.1, 77.2, 67.3, 54.5, 47.2, 38.0, 25.0. Total yield=8.238 g, 16.0mmoles, 62.6% overall yield.

Fmoc-protected boronated amino acids as described herein are employed toprepare boronated peptides for boronation of peptides and proteins. Forexample, Fmoc-protected boronated glutamic acid is used in place ofFmoc-protected glutamic acid in conventional Fmoc solid phase peptidesynthesis to prepare boronated oligopeptides containing one or moreboronated glutamic acids, such asAnx-GE_(B)GE_(B)GE_(B)GE_(B)GE_(B)GE_(B)G, where E_(B) representboronated glutamic acid. Similarly, Fmoc-protected boronatedphenylalanine can be used to prepare boronated oliogpeptides containingone or more boronated phenylalanines. Conventional Fmoc solid phasedescribed in Chan, W. C. and White P. D. Fmoc Solid Phase PeptideSynthesis-A Practical Approach, Oxford University Press 2000. Thisreference is incorporated by reference herein in its entirety for itsdescription of methods and materials for solid phase peptide synthesis.

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1. A method for increasing cellular uptake of a cargo molecule byboronating the cargo molecule by binding one or more phenylboronatecompounds to the cargo molecule.
 2. The method of claim 1 wherein theone or more phenylboronate compounds are selected from those havingformulas:

or salts thereof, where: x is 1 or 2 R₁ and R₅ are independentlyselected from hydrogen, an optionally substituted straight-chain orbranched aliphatic group having 1-8 carbon atoms, a —CO₂R₁₀ group, a—OCO—R₁₀ group, a —CON(R₁₂)₂ group, a —N(R₁₂)₂ group, a —OR₁₀ group, a—(CH₂)₂—OR₁₀ group, a —(CH₂)₂—N(R₁₂)₂ group, a halogen, a nitro group,or a cyano group; R₂, R₃, R₄ and R₆ are independently selected fromhydrogen, a straight-chain or branched aliphatic group having 1-8 carbonatoms, an alicyclic group, an aryl group, a heterocyclic group, aheteroaryl group, a —CO₂R₁₀ group, a —OCO—R₁₀ group, a —CON(R₁₂)₂ group,a —N(R₁₂)₂ group, a —OR₁₀ group, a —(CH₂)_(m)OH group, a halogen, anitro group, a cyano group, -M, or two adjacent R₂-R₆, together with thering carbons to which they are attached, optionally form a 5-8-memberalicyclic, heterocyclic, aryl or heteroaryl ring moiety, each of whichgroups or moieties is optionally substituted, wherein: each R₁₀ isindependently selected from hydrogen, a straight-chain or branchedaliphatic group having 1-8 carbon atoms, an alicyclic group, an arylgroup, a heterocyclic group, or a heteroaryl group, each of which groupsis optionally substituted; each R₁₂ is independently selected fromhydrogen, a straight-chain or branched aliphatic group having 1-8 carbonatoms, an alicyclic group, an aryl group, a heterocyclic group, aheteroaryl group, or where two R₁₂ together with the nitrogen to whichthey are attached can form a 5-8 member heterocyclic or heteroaryl ringmoiety, each of which groups or moieties is optionally substituted; m isan integer from 1-8; and M is a reactive group or a linker carrying areactive group, wherein the reactive group is functional for covalentattachment of the phenylboronate to a peptide or protein and which: (1)reacts with one or more of: an amine group, a carboxylic acid group, asulfhydryl group or a hydroxyl group, particularly where that group is agroup of a natural or unnatural amino acid of such an amino acid of apeptide or a protein; (2) reacts with an aldehyde or ketone group, anazide group, an activated ester group, a thioester group,phosphinothioester, or other group which is introduced into or generatedin the amino acid, peptide or protein; or (3) reacts with one reactivegroup of a homobifunctional or a heterobifunctional crosslinkingreagent, wherein at least one of R₁-R₆ is M and wherein optionalsubstitution is substitution by one or more substituents selected fromhalogen; a nitro group; a cyano group; a C1-C6 alkyl group; a C1-C6alkoxy group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a 3-7 memberalicyclic ring, wherein one or two ring carbons are optionally replacedwith —CO— and which may contain one or two double bonds; an aryl grouphaving 6-14 carbon ring atoms; a phenyl group; a benzyl group; a 5- or6-member ring heterocyclic group having 1-3 heteroatoms and wherein aring carbon is optionally replaced with —CO— and which may contain oneor two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, Oor S); a —CO₂R₁₃ group; —OCO—R₁₃ group; —CON(R₁₄)₂ group; —N(R₁₄)₂group; or —OR₁₃ group, where each R₁₃ or R₁₄ is independently hydrogen;an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having6-14 carbon atoms; an unsubstituted phenyl group; an unsubstitutedbenzyl group; an unsubstituted 5- or 6-member ring heterocyclic grouphaving 1-3 heteroatoms and wherein a ring carbon is optionally replacedwith —CO— and which may contain one or two double bonds; or a heteroarylgroup having 1-3 heteroatoms (N, O or S) and in addition two R₁₄together with the nitrogen to which they are attached can form aheterocyclic or heteroaryl ring moiety, each of which groups or moietiesis optionally substituted; each of which R₁₃ and R₁₄ groups is in turnoptionally substituted with one or more unsubstituted C1-C3 alkylgroups, halogens, nitro groups, cyano groups, —CO₂R₁₅ groups, —OCO—R₁₅groups, —CON(R₁₆)₂ groups, —N(R₁₆)₂ groups, or —OR₁₅ groups, where eachof R₁₅ and R₁₆ independently are hydrogen, an unsubstituted C1-C6 alkylgroup; an unsubstituted aryl group having 6-14 carbon ring atoms; anunsubstituted phenyl group; an unsubstituted benzyl group, anunsubstituted 5- or 6-member ring heterocyclic group having 1-3heteroatoms and wherein a ring carbon is optionally replaced with —CO—and which may contain one or two double bonds; or a heteroaryl grouphaving 1-3 heteroatoms (N, O or S) and a total of 5-14 ring atoms; andin addition two R₁₆ together with the nitrogen to which they areattached can form an unsubstituted heterocyclic or heteroaryl ringmoiety.
 3. The method of claim 2 wherein M is a reactive group or alinker containing a reactive group wherein the reactive group reactswith one or more of: an amine, a carboxylic acid, a sulfhydryl group ora hydroxyl group.
 4. The method of claim 2 wherein M is or contains anamine-reactive functional group.
 5. The method of claim 2 wherein M isor contains a carboxyl-reactive functional group.
 6. The method of claim2 wherein M is or contains a sulfhydryl-reactive functional group. 7.The method of claim 2 wherein M is or contains a hydroxyl-reactivefunctional group.
 8. The method of claim 2 wherein M is or contains areactive group which reacts with an aldehyde or ketone group, an azidegroup, an activated ester group, a thioester group, or other group whichis introduced into the peptide or protein by reaction of a convertingreagent with one or more of an amine group, a carboxylic acid group, asulfhydryl group or a hydroxyl group.
 9. The method of claim 1 whereinthe cargo molecule is a nucleic acid, a peptide or a protein.
 10. Themethod of claim 1 wherein the cargo molecule is an enzyme, an antibodyor a functional fragment thereof.
 11. The method of claim 1 wherein theone or more phenylboronate compounds are bound to the cargo molecule bybinding of a phenylboronated oligopeptide to the peptide or protein. 12.The method of claim 1 wherein one or more phenylboronate compounds orone or more phenylboronated oligopeptides are ligated, crosslinked orotherwise attached to the cargo molecule.
 13. A cargo molecule to whichone or more phenylboronate moieties are covalently bound.
 14. The cargomolecule of claim 13 which is a peptide, a protein or a nucleic acid.15. The cargo molecule of claim 13 wherein the one or morephenylboronate moieties are selected from those having formulas:

or salts thereof, where: x is 1 or 2; R₁ and R₅ are independentlyselected from hydrogen, an optionally substituted straight-chain orbranched aliphatic group having 1-8 carbon atoms, a —CO₂R₁₀ group, a—OCO— R₁₀ group, a —CON(R₁₂)₂ group, a —N(R₁₂)₂ group, a —OR₁₀ group, a—(CH₂)₂—OR₁₀ group, a —(CH₂)₂—N(R₁₂)₂ group, a halogen, a nitro group,or a cyano group; R₂, R₃, R₄ and R₆ are independently selected fromhydrogen, a straight-chain or branched aliphatic group having 1-8 carbonatoms, an alicyclic group, an aryl group, a heterocyclic group, aheteroaryl group, a —CO₂R₁₀ group, a —OCO—R₁₀ group, a —CON(R₁₂)₂ group,a —N(R₁₂)₂ group, a —OR₁₀ group, a —(CH₂)_(m)OH group, a halogen, anitro group, a cyano group, -L-, or two adjacent R₂-R₆, together withthe ring carbons to which they are attached, optionally form a5-8-member alicyclic, heterocyclic, aryl or heteroaryl ring moiety, eachof which groups or moieties is optionally substituted, wherein: each R₁₀is independently selected from hydrogen, a straight-chain or branchedaliphatic group having 1-8 carbon atoms, an alicyclic group, an arylgroup, a heterocyclic group, or a heteroaryl group, each of which groupsis optionally substituted; each R₁₂ is independently selected fromhydrogen, a straight-chain or branched aliphatic group having 1-8 carbonatoms, an alicyclic group, an aryl group, a heterocyclic group, aheteroaryl group, or where two R₁₂ together with the nitrogen to whichthey are attached can form a 5-8 member heterocyclic or heteroaryl ringmoiety, each of which groups or moieties is optionally substituted; m isan integer from 1-8; and -L- is a linker through which thephenylboronate is covalently bonded to the peptide or protein; whereinat least one of R₁-R₆ is -L- and wherein optional substitution issubstitution by one or more substituents selected from halogen; a nitrogroup; a cyano group; a C1-C6 alkyl group; a C1-C6 alkoxy group; a C2-C6alkenyl group; a C2-C6 alkynyl group; a 3-7 member alicyclic ring,wherein one or two ring carbons are optionally replaced with —CO— andwhich may contain one or two double bonds; an aryl group having 6-14carbon ring atoms; a phenyl group; a benzyl group; a 5- or 6- memberring heterocyclic group having 1-3 heteroatoms and wherein a ring carbonis optionally replaced with —CO— and which may contain one or two doublebonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S); a—CO₂R₁₃ group; —OCO—R₁₃ group; —CON(R₁₄)₂ group; —N(R₁₄)₂ group; or—OR₁₃ group, where each R₁₃ or R₁₄ is independently hydrogen; anunsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14carbon atoms; an unsubstituted phenyl group; an unsubstituted benzylgroup; an unsubstituted 5- or 6-member ring heterocyclic group having1-3 heteroatoms and wherein a ring carbon is optionally replaced with—CO— and which may contain one or two double bonds; or a heteroarylgroup having 1-3 heteroatoms (N, O or S) and in addition two R₁₄together with the nitrogen to which they are attached can form aheterocyclic or heteroaryl ring moiety, each of which groups or moietiesis optionally substituted; each of which R₁₃ and R₁₄ groups is in turnoptionally substituted with one or more unsubstituted C1-C3 alkylgroups, halogens, nitro groups, cyano groups, —CO₂R₁₅ groups, —OCO—R₁₅groups, —CON(R₁₆)₂ groups, —N(R₁₆)₂ groups, or —OR₁₅ groups, where eachof R₁₅ and R₁₆ independently are hydrogen, an unsubstituted C1-C6 alkylgroup; an unsubstituted aryl group having 6-14 carbon ring atoms; anunsubstituted phenyl group; an unsubstituted benzyl group, anunsubstituted 5- or 6-member ring heterocyclic group having 1-3heteroatoms and wherein a ring carbon is optionally replaced with —CO—and which may contain one or two double bonds; or a heteroaryl grouphaving 1-3 heteroatoms (N, O or S) and a total of 5-14 ring atoms; andin addition two R₁₆ together with the nitrogen to which they areattached can form an unsubstituted heterocyclic or heteroaryl ringmoiety. 16-17. (canceled)
 18. A boronation reagent of formula:

or salts thereof, where: M2 is a spacer substituted with a reactivegroup and which contains a latent reactive group; x is 1 or 2; R₁ isselected from hydrogen, an optionally substituted straight-chain orbranched aliphatic group having 1-8 carbon atoms, a —CO₂R₁₀ group, a—OCO—R₁₀ group, a —CON(R₁₂)₂ group, a —N(R₁₂)₂ group, a —OR₁₀ group, a—(CH₂)₂—OR₁₀ group, a —(CH₂)₂—N(R₁₂)₂ group, a halogen, a nitro group,or a cyano group; R₂, R₃, and R₄ are independently selected fromhydrogen, a straight-chain or branched aliphatic group having 1-8 carbonatoms, an alicyclic group, an aryl group, a heterocyclic group, aheteroaryl group, a —CO₂R₁₀ group, a —OCO—R₁₀ group, a —CON(R₁₂)₂ group,a —N(R₁₂)₂ group, a —OR₁₀ group, a —(CH₂)_(m)OH group, a halogen, anitro group, a cyano group, or two adjacent R₂-R₆, together with thering carbons to which they are attached, optionally form a 5-8-memberalicyclic, heterocyclic, aryl or heteroaryl ring moiety, each of whichgroups or moieties is optionally substituted, wherein: each R₁₀ isindependently selected from hydrogen, a straight-chain or branchedaliphatic group having 1-8 carbon atoms, an alicyclic group, an arylgroup, a heterocyclic group, or a heteroaryl group, each of which groupsis optionally substituted; each R₁₂ is independently selected fromhydrogen, a straight-chain or branched aliphatic group having 1-8 carbonatoms, an alicyclic group, an aryl group, a heterocyclic group, aheteroaryl group, or where two R₁₂ together with the nitrogen to whichthey are attached can form a 5-8 member heterocyclic or heteroaryl ringmoiety, each of which groups or moieties is optionally substituted; andm is an integer from 1-8; R₇ and R₈ are selected from a hydrogen or anoptionally substituted C1-C3 alkyl group; —O—Y is —O—CO—R₂₄, where R₂₄is an optionally substituted alkyl or aryl group, a —O—PO₃H₂ or acorresponding phosphate ester thereof or a —O—SO₂—R₂₅ group, where R₂₅is an optionally substituted alkyl or aryl group; and wherein optionalsubstitution is substitution by one or more substituents selected fromhalogen; a nitro group; a cyano group; a C1-C6 alkyl group; a C1-C6alkoxy group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a 3-7 memberalicyclic ring, wherein one or two ring carbons are optionally replacedwith —CO— and which may contain one or two double bonds; an aryl grouphaving 6-14 carbon ring atoms; a phenyl group; a benzyl group; a 5- or6-member ring heterocyclic group having 1-3 heteroatoms and wherein aring carbon is optionally replaced with —CO— and which may contain oneor two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, Oor S); a —CO₂R₁₃ group; —OCO—R₁₃ group; —CON(R₁₄)₂ group; —N(R₁₄)₂group; or —OR₁₃ group, where each R₁₃ or R₁₄ is independently hydrogen;an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having6-14 carbon atoms; an unsubstituted phenyl group; an unsubstitutedbenzyl group; an unsubstituted 5- or 6-member ring heterocyclic grouphaving 1-3 heteroatoms and wherein a ring carbon is optionally replacedwith —CO— and which may contain one or two double bonds; or a heteroarylgroup having 1-3 heteroatoms (N, O or S) and in addition two R₁₄together with the nitrogen to which they are attached can form aheterocyclic or heteroaryl ring moiety, each of which groups or moietiesis optionally substituted; each of which R₁₃ and R₁₄ groups is in turnoptionally substituted with one or more unsubstituted C1-C3 alkylgroups, halogens, nitro groups, cyano groups, —CO₂R₁₅ groups, —OCO—R₁₅groups, —CON(R₁₆)₂ groups, —N(R₁₆)₂ groups, or —OR₁₅ groups, where eachof R₁₅ and R₁₆ independently are hydrogen, an unsubstituted C1-C6 alkylgroup; an unsubstituted aryl group having 6-14 carbon ring atoms; anunsubstituted phenyl group; an unsubstituted benzyl group, anunsubstituted 5- or 6-member ring heterocyclic group having 1-3heteroatoms and wherein a ring carbon is optionally replaced with —CO—and which may contain one or two double bonds; or a heteroaryl grouphaving 1-3 heteroatoms (N, O or S) and a total of 5-14 ring atoms; andin addition two R₁₆ together with the nitrogen to which they areattached can form an unsubstituted heterocyclic or heteroaryl ringmoiety; with the proviso that in formula F1 one of R₂ or R₃ is —B(OH)₂or R₂ and R₃ together are:


19. The boronation reagent of claim 18 of formula:

or salts thereof; where: R₃₀ and R₃₁ are H, alkyl or R₃₀ and R₃₁together with the intervening C═C moiety form a 6-8 member carbocyclicor heterocyclic ring wherein one of the ring carbons can be replacedwith —CO—; and X—CO— is an activated ester; with the proviso that informula II A, one of R₂ or R₃ is —B(OH)₂ or R₂ and R₃ together are:


20. The boronation reagent of claim 18 of formula:

or salts thereof wherein X—CO— is an activated ester; with the provisothat in formula IVA one of R₂ or R₃ is —B(OH)₂ or R₂ and R₃ togetherare:


21. The boronation reagent of claim 19 wherein R₃₀ and R₃₁ are H, oralkyl groups having 1-3 carbon atoms and —COX is an activated esterselected from N-hydroxysuccinimide esters or N-hydroxysulfosuccinimideesters.
 22. The boronation reagent of claim 20 wherein R₃₀ and R₃₁ areH, or alkyl groups having 1-3 carbon atoms and —COX is an activatedester selected from N-hydroxysuccinimide esters orN-hydroxysulfosuccinimide esters.